Bio-Rad S3 User Manual

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S3™ and S3e™ Cell Sorters

Instruction Manual

Catalog #145-1001 #145 -1002 #14 5 -10 05 #14 5 -10 0 6 #145-1008

Bio-Rad Technical Support

For help and technical advice, please contact the Bio-Rad Technical Support department. In the United States, the Technical Support department is open Monday–Friday, 5:00 AM–5:00 PM, Paciﬁc time.

http://www.bio-rad.com

Bio-Rad Laboratories Life Science Research 2000 Alfred Nobel Drive Hercules, CA 94547

Telephone: 510-741-1000 Tele x: 335-358

Toll Fre e: 1-800-4-BIORAD (1-800-424-6723) Fax: 510-741-5800 Free Fax: 1-800-879-2289

Online technical support and worldwide contact information are available at www.consult.bio-rad.com.

Legal Notices

Agilis is a trademark of Newport Corporation.

Kimwipes is a trademark of Kimberly-Clark Corporation.

Windows is a trademark of Microsoft Corporation.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage or retrieval system, without permission in writing from Bio-Rad Laboratories.

Bio-Rad reserves the right to modify its products and services at any time. This instruction manual is subject to change without notice.

Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors, or for any damages resulting from the application or use of this information.

Instruction Manual | i

Bio-Rad Laboratories Resources

Bio-Rad provides many resources for scientists. Table 1 lists available resources and how to locate what you need.

Table 1. Bio-Rad resources.

Resource How to Contact

Local Bio-Rad Laboratories Find local information and contacts on the Bio-Rad Laboratories website by selecting your country representatives on the homepage (www.bio-rad.com). Find the nearest international ofﬁce listed on the back of

this manual

Technical support scientists Bio-Rad’s technical support scientists provide our customer with practical and expert solutions.

To ﬁnd local technical support on the phone, contact your nearest Bio-Rad ofﬁce. For technical support in the United States and Canada, call 1-800-424-6723 (toll-free), and select the technical support option

Service support engineers Maintenance and repairs should be carried out only by authorized service support engineers

For service support in the United States and Canada, call 1-800-424-6723 (toll-free), and select

the technical support option to request service support

Technical notes and literature Go to the Bio-Rad website (www.bio-rad.com). Type a term in the Search box and select

Documents tab to ﬁnd links to literature

Writing Conventions Used in This Manual

This manual uses the writing conventions listed in Table 2.

Table 2. Conventions used in this manual.

Convention Meaning

Note: Provides helpful information and instructions, including information explained in further detail elsewhere in this manual

WARNING! Explains very important information about something that might injure the researcher, damage the instrument, or

cause data loss

X > Y Instruction to select X and then select Y from a toolbar, menu, or software window

Highlights area of interest on a screenshot

IMPORTANT! Provides important information about necessary actions or common mistakes

ii | S3 and S3e Cell Sorters

Safety and Regulatory Compliance

For safe operation of the S3 and S3e Cell Sorter systems, we strongly recommend that you follow the safety speciﬁcations listed in this section and throughout the manual.

Safety Warning Labels

Warning labels posted on the instrument and in this manual warn you about sources of injury or harm. Refer to Table 3 to review the meaning of each safety warning label.

Table 3. Meaning of safety warning labels.

CAUTION: Shock hazard! This symbol draws attention to a possible injury or danger to life if the associated

directions are not followed correctly

CAUTION: Risk of danger! This symbol identiﬁes components that pose a risk of personal injury or damage to

the instrument if improperly handled. Wherever this symbol appears, consult the manual for further information before proceeding

CAUTION: Laser hazard! This symbol draws attention to a possible injury or danger to life due to laser radiation

if the associated directions are not followed correctly

CAUTION: Biohazard! This symbol identiﬁes components that may become contaminated with biohazardous material

Instrument Warning Labels

The warning labels shown in Table 4 are displayed on the instrument and refer directly to the safe use of the S3 and S3e Cell Sorter systems.

Table 4. Instrument safety warning labels.

Warning about risk of shock.

Only qualiﬁed, trained technicians should carry out service work on electronic components due to potential shock hazard

Warning about electronic components.

Electronic components are sensitive to electrostatic charges and can be destroyed by a discharge

Warning about weight of the system.

Lifting should be accomplished with a minimum of two people and only by the inset handles on the instrument base. Use caution to keep instrument level and handle gently

Warning about handling biohazardous materials.

When handling biohazardous samples or the S3 or S3e System’s waste container, adhere to the recommended precautions and guidelines in this manual, and comply with any local guidelines speciﬁc to your laboratory and location

Instruction Manual | iii

Safe Use Speciﬁcations and Compliance

Laser Product Hazard Classiﬁcation

The intent of the laser hazard classiﬁcation is to identify hazards to users posed by the laser, and provide appropriate protective measures. The S3 or S3e laser is a Class 1 laser product that complies with 21 CFR 1040.10 and 1040.11, except for deviations pursuant to Laser Notice No. 50, dated June 24, 2007 stating that operators are not exposed to harmful levels of laser radiation during normal operation, maintenance and/or service. During times of repair and/or major service by a trained technician, laser safety controls for Class 3B lasers must be followed.

WARNING! Use of controls or adjustments or performance of procedures other than those speciﬁed herein may result in hazardous laser radiation exposure.

Electrical Safety Information and Classiﬁcation

The S3 and S3e Systems conform to international regulations encompassing the accessibility of high voltages by the user. Use all protective housings, interlocks, and shields as identiﬁed in this manual. Further information about speciﬁc electrical hazards is listed in the hardware description.

AC Fuse Requirements

Remove power cord before replacing fuses. Fuses are 5 x 20 mm and must be rated to 250 VAC, 4 A slow blow such as Schurter 0034.3123

AC Power Cord Requirements

Power cord must be IEC 60320-1 compliant with a C13 plug on the instrument end. The power cord must be rated at minimum 250 VAC, 10 A at 60ºC minimum. In the U.S. and Canada, the power cord must be rated at minimum 125 VAC, 10 A at 60ºC minimum.

Position the instrument for easy access to the power switch and the power cord.

Regulatory Compliance

This instrument has been tested and found to be in compliance with all applicable requirements of the following safety and electromagnetic standards:

IEC 61010-1:2010 (3rd Ed.), EN61010-1:2010 (3rd Ed). Electrical Equipment for Measurement, Control, and Laboratory Use - Part 1:

General Requirements.

UL/CSA 61010-1:2012 (3rd Ed.), Standard for Safety Electrical Equipment for Electrical Safety (USA, Canda, NRTL)

IEC 60825-1:2007(2nd Ed.), EN 60825-1:2007(2nd Ed). Safety of laser products - Part 1: Equipment classiﬁcation and requirements

Class 1 laser product per CDRH requirements and regulations

IEC 61010-2-081:2001+A1, EN61010-2-081:2002+A1. Safety requirements for electrical equipment for measurement, control and

laboratory use. Part 2-081: Particular requirements for automatic and semi-automatic laboratory equipment for analysis and other purposes (includes Amendment 1)

EN 61326-1:2006 (Class A) Electrical equipment for measurement, control and laboratory use. EMC requirements, Part 1: General

requirements

This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference, in which case the user will be required to correct the interference at his own expense.

Hazards

The S3 and S3e Cell Sorters are designed to operate safely when used in the manner prescribed by the manufacturer. If the S3 or S3e Cell Sorter or any of its associated components are used in a manner other than prescribed, or if modiﬁcations to the instrument are not performed by a Bio-Rad or other authorized agent, then the warranty on the system will be voided. Service of the S3 or S3e Cell Sorter should be performed only by Bio-Rad personnel.

Biohazards

The S3 and S3e Cell Sorters are laboratory products. However, if biohazardous samples are present, adhere to the following guidelines and comply with any local guidelines speciﬁc to your laboratory and location.

General Precautions

Always wear laboratory gloves, coats, and safety glasses with side shields or goggles

Keep your hands away from your mouth, nose, and eyes

Completely protect any cut or abrasion before working with potentially infectious materials

iv | S3 and S3e Cell Sorters

Wash your hands thoroughly with soap and water after working with any potentially infectious material before leaving the laboratory

Remove wristwatches and jewelry before working at the bench

Store all infectious or potentially infectious material in unbreakable leak-proof containers

Before leaving the laboratory, remove protective clothing

Do not use a gloved hand to write, answer the telephone, turn on a light switch, or touch anything that other people may touch

without gloves

Change gloves frequently. Remove gloves immediately when they are visibly contaminated

Do not expose materials that cannot be properly decontaminated to potentially infectious material

Upon completion of the operation involving biohazardous material, decontaminate the work area with an appropriate disinfectant

(for example, a 1:10 dilution of household bleach)

No biohazardous substances are exhausted during normal operations of this instrument

Disposal of Biohazardous Material

The S3 and S3e Systems include a waste container that may potentially contain hazardous biological materials, depending on the sample used. Dispose of the following potentially contaminated materials in accordance with laboratory, local, regional, and national regulations:

Content in waste container

Reagents

Used reaction vessels or other consumables that may be contaminated

Chemical Hazards

The S3 and S3e Systems include a waste container that may potentially contain hazardous chemical materials depending on the sample used.

Explosive or Flammability Hazards

The S3 and S3e Systems pose no uncommon hazard related to ﬂammability or explosion when used in a proper manner as speciﬁed by Bio-Rad Laboratories.

Electrical Hazards

The S3 and S3e Systems pose no uncommon electrical hazard to operators if installed and operated properly without physical modiﬁcation and if connected to a power source of proper speciﬁcation.

Transport

Moving the S3 and S3e Systems is not recommended after installation. If the system needs to be moved, follow the decontamination procedure in this manual and remove all bulk ﬂuidics. A QC procedure will be required after a move to ensure instrument is functioning properly.

Lifting should be performed with a minimum of two people. Lift with the inset handles on the instrument base. Use caution to keep instrument level, and handle the instrument gently.

Storage

The S3 or S3e System can be stored under the following conditions:

Temperature range 5–35°C

Relative humidity 20–70%

Disposal

The S3 and S3e Systems contain electronic or electrical materials; it should be disposed of as unsorted waste and must be collected separately, according to European Union Directive 2002/96/CE on waste and electronic equipment — WEEE Directive. Before disposal, contact your local Bio-Rad representative for country-speciﬁc instructions.

Warranty

The S3 and S3e Cell Sorters and associated accessories are covered by a standard Bio-Rad warranty. Contact your local Bio-Rad Laboratories ofﬁce for details of the warranty.

Instruction Manual | v

vi | S3 and S3e Cell Sorters

Table of Contents

Chapter 1: Introduction 1

1.1 System Components 2

1.2 Installation Requirements 2

Chapter 2: Hardware Description 3

2.1 System Overview 3

2.2 Fluidics System 5

2.3 Optics 9

2.4 Electronics 11

Chapter 3: ProSort™ So ftwar e 13

3.1 Main Software Window 14

3.2 Control Panel 20

3.3 Administrator Tab Toolbar 22

Chapter 4: Getting Started 33

4.1 Checking Bulk Fluidics 33

4.2 Logging In 34

4.3 Daily Startup 35

4.4 Quality Control 36

4.5 Protocols and Workspace 42

Chapter 5: Acquisition 51

5.1 Acquisition Setup 51

5.2 Compensation 55

5.3 Checking or Swapping Fluidics 60

5.4 Optical Filters 62

Chapter 7: Additional Software Features 73

7.1 Debubble 73

7.2 Unclog 73

7.3 Swap Tip 73

7.4 Clean System 74

7.5 Instrument Status Box 75

7.6 Status Bar 75

7.7 Printing 76

7.8 Quality Control Reports 76

7.9 User Reports 77

7.10 Biosafety System 78

Chapter 8: Shutdown 79

8.1 Daily Shutdown 79

Chapter 9: Automatic Startup 83

9.1 Scheduling an Automatic Startup 83

9.2 Previously Scheduled Automatic Startups 84

Chapter 10: Maintenance 87

10.1 General Maintenance 87

10.2 Dealing with Clogs 88

10.3 Cleaning or Replacing Nozzle Tip 88

10.4 Swap Nozzle Tip Wizard 89

10.5 Optical Filter Cleaning 105

10.6 Disinfectants 105

10.7 Decontamination 105

Chapter 6: Sorting 65

6.1 Sort Setup 65

6.2 Sort Modes 69

6.3 Sort Statistics 69

6.4 Sort Plots 71

Troubleshooting 111

References 115

S3 and S3e Cell Sorter Specifications 117

Ordering Information 119

Instruction Manual | vii

viii | S3 and S3e Cell Sorters

Introduction

The S3™ and S3e™ Cell Sorters are designed to offer affordable, dependable, and simpliﬁed cell sorting. As benchtop cell sorters, the S3 and S3e Cell Sorters are equipped with one or two lasers and up to four ﬂuorescent detectors, plus forward and side scatters. Samples are analyzed using the traditional jet-in-air technology, and events can be sorted at high speeds while maintaining sensitivity and high purity.

Sorting is typically accomplished by breaking the stream of ﬂuid containing particles into droplets and applying a charge to the stream when a particle passes the criteria to be sorted. The charged droplet containing the particle is then deﬂected by an electric ﬁeld into a collection vessel. To ensure proper deﬂection of the correct droplets, proper instrument setup is integral to achieving high-purity sort results.

By automating the complex presort setup with a unique technology, the S3 and S3e create a more efﬁcient and consistent workﬂow.

Key automated features:

Startup/shutdown

Stream-to-laser alignment

Drop delay calculation

Droplet break-off monitoring and feedback

Phase and deﬂection adjustments to sort stream

Daily QC reporting and trending

Collection volume monitoring to prevent overﬁlling

The S3 and S3e system. The instrument uses a unique internal buffer chamber system for dilution of 8x sheath ﬂuid with deioinized (DI) water, allowing users to swap ﬂuids without the need to shut down the system. This process is known as a “hot swap,” and allows uninterrupted sorting. For more information, refer to Section 5.3, Checking or Swapping Fluidics.

Cell Sorters include a complete internalized ﬂuidics and temperature control

Cell Sorters

Instruction Manual | 1

Introduction

1.1 System Components

The S3 or S3e Cell Sorter complete system includes the following components:

S3 or S3e Cell Sorter Instrument, 1 each

n

S3 or S3e Installation Kit, 1 each

S3 cap assembly, white, sheath ﬂuid, 1 each S3 cap assembly, blue, DI water, 1 each S3 cap assembly, red, waste, 1 each S3 ﬂuidics container 4 L size, empty, 2 each S3 individual ﬁlter holder, black, empty, 5 each S3 forward scatter (FSC) ﬁlter holder

with neutral density ﬁlter, 2.0, 1 each S3 ﬁlter block A with preset ﬁlter

conﬁguration, 1 each S3 ﬁlter block B with preset ﬁlter

conﬁguration, 1 each

S3 Accessory Kit (optional), 1 each (catalog #145-1065)

S3 nozzle tip, 100 µm, 1 each S3 nozzle O-rings, 2 each S3 nozzle alignment disk, 2 each Neutral density ﬁlter, 1.0, 2 each

5 ml tubes, 12 x 75 mm, 1 pack S3 collection adaptor set, 1 each Bulk ﬂuidics tray, 3 each USB cord, 1 each Ethernet cord, 1 each Power cord, 1 each USB drive with ProSort

™

software Instrument and software manual Instrument quick guide

2 mm hex driver, 1 each S3 spanner wrench, 1 each 1 ml syringe, 1 each Plastic box, 1 each

Computer CPU and 24 inch monitor, 1 each (catalog #145-1066)

ProFlow

ProLine

™

8x Sheath Fluid, preservative free, 5 x 4 L, 1 case (catalog #145-1082)

™

Calibration Beads, 3 x 5 ml, 1 pack (catalog #145-1081) or

ProLine Universal Calibration Beads, 3 x 5 ml, 1 pack (catalog #145-1086)

1.2 Installation Requirements

The S3 and S3e Cell Sorters should be installed by a trained service engineer to ensure proper operation and calibration of the instrument. If any items are missing or damaged, contact your local Bio-Rad ofﬁce for assistance.

Before the S3 and S3e chosen. The instrument should be located on a sturdy bench or table top, away from any other instruments that may interfere electrically or mechanically by causing vibration. The bench or table top must be able to accommodate 198 lb (90 kg), the weight of the instrument. The area should be free of excessive dust or moisture.

Table 5. Dimensions for instrument alone or with computer and monitor.*

Instrument only (W x D x H) 70 x 65 x 65 cm

Instrument wiith computer and monitor, 116 x 65 x 65 cm (W x D x H) 46 x 25.5 x 25.5 in

* An additional 61 cm (24 in) of height clearance is needed for service.

Cell Sorters can be installed by a service engineer, a site must be

27.5 x 25.5 x 25.5 in

2 | S3 and S3e Cell Sorters

Hardware Description

This chapter describes the hardware of the S3™ and S3e™ systems. Understanding the system’s hardware is essential for proper operation.

2.1 System Overview

The S3 and S3e Systems consist of ﬂuidics, optics, electronics, and software. These can be broken down into several subsystems (Figure 1).

Access to nozzle assembly

Access to internal ﬂuidics system

Fig. 1. Front view of the S3 system.

Access to ﬁlters

Touch locking system

Sort collection area

Loading stage

Instruction Manual | 3

Hardware Description

2.1.1 Instrument Back Panel

The rear connector panel of the S3 or S3e system includes these features:

n

Main power switch (black) — press the main power switch to turn on power to the system.

WARNING! The main power switch should not be used to shut down the system. Perform system shutdown from the ProSort™ software. For more information, refer to Chapter 8, Shutdown

n

Power input (black) — plug in the power cord here. The system requires 100 or 240 VAC outlet

n

USB port (gray) — use this port to connect the system to the computer for communication

n

Ethernet port (green) — use this port to connect the system to the computer for communication

There is a color-coded guide for proper connection location and orientation (Figure 2).

Fig. 2. Instrument rear connector panel.

CAUTION: Three cords connect the instrument to the computer for power and communication. Be cautious when walking around the instrument, as these cords can become a tripping hazard.

2.1.2 Aerosol Evacuation Port

The S3 and S3e systems also include an aerosol evacuation port used to directly evacuate the sort collection chamber when connected to a biosafety system. This port can be covered when not in use.

Fig. 3. Aerosol evacuation port.

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2.2 Fluidics System

The S3 and S3e ﬂuidics system consists of the bulk ﬂuidics, loading stage, nozzle, and sort collection chamber. The ﬂuidics system supplies sheath ﬂuid, DI water, and sample to the nozzle, and then collects the waste for proper disposal.

CAUTION: Biohazard! Biosafety is of utmost importance while operating this instrument. Consult with your local safety ofﬁcer or review local, state, and federal regulations to ensure proper handling and disposal of biohazardous substances.

2.2.1 Bulk Fluidics

The S3 and S3e system includes two empty containers as part of the bulk ﬂuidics. Table 6 describes the function of each container.

Table 6. Containers and their functions.

Label Container Function

Biohazardous The waste container holds the system’s ﬂuid after it has run through the nozzle

Waste and waste lines. This container has a red cap and holds 4 liters of ﬂuids.

Sheath Fluid The sheath ﬂuid container holds the 8x sheath ﬂuid for the system. This container

Deionized water The DI water container holds deionized water for mixing with the 8x sheath ﬂuid to (DI water) create a 1x sheath ﬂuid. This container has a blue cap and holds 4 liters of DI

2.2 Fluidics System

Approximately 9 hours of run time can be performed until the empty container is ﬁlled. The ﬂuid collected in this container should be decontaminated as appropriate to the application and samples being run on the instrument. Please verify proper treatment and disposal with your safety ofﬁcer or local health and safety bodies

has a white cap and holds 4 liters of ﬂuid. If preferred, 1x sheath ﬂuid may be used instead. In this case, the DI water container will not be used to dilute the sheath, but only for rinsing and cleaning. If 1x sheath ﬂuid is used, ensure the option is checked in the Global Preferences of the software. This setting will apply globally to the system when set by an administrator. A full container of 8x sheath ﬂuid has approximately 50 hours of run time, while a full tank of 1x sheath ﬂuid has approximately 9 hours of run time

water with a run time of approximately 9 hours between reﬁlls when using 8x sheath ﬂuid. The DI water is also used for cleaning at the end of the day and to rinse the sample probe between samples

Note: The ProFlow™ 8x sheath ﬂuid is diluted with the DI water using an internal ﬂuidics chamber.

Each container uses a quick disconnect

Sheath ﬂuid cap assembly

Sheath ﬂuid container

system for easy swapping (Figure 4). Both the DI water and the sheath ﬂuid containers have one connector, while the waste container has two connectors for waste ﬂuid from the sorting chamber and the washing station. Located above the containers inside the instrument is a magnetic holder for each quick disconnect. The magnetic holder will keep the quick disconnect away from exchange area.

Fig. 4. Fluidics container highlighting the quick disconnect system for the sheath ﬂuid container.

Instruction Manual | 5

Quick disconnect

Hardware Description

The sheath ﬂuid and DI water are both ﬁltered through a 0.2 µm ﬁlter to remove any particulates from the ﬂuid before circulating through the system. Fluidic levels are monitored by the software using a weight measurement system. Below each ﬂuidic container is a bulk ﬂuidics tray. Each tray contains a sensor that translates volume weight into ﬂuidic run time and helps to keep the system dry while swapping ﬂuidics.

IMPORTANT! Replace ﬁlter cartridges on a regular basis. Filter replacement is part

of the annual service preventative maintenance visit. For additional information, refer to Chapter 10, Maintenance.

2.2.2 Loading Stage

The loading stage consists of two functional stations; sample input and washing. The sample input station is located in the front position of the loading stage and it supports a 5 ml, 12 x 75 mm tube (Figure 5).

Note: Polypopylene tubes for sample acquisition are recommended, but not required.

When a sample is loaded and ready to run, the loading stage can be moved into the run position.

To move the loading stage into the run position:

1. Push the handle on the loading stage down.

2. Push the loading stage inward.

3. Gently raise the loading stage into the run position.

The locking mechanism automatically engages when the tube is in the run position (Figure 6). After the tube is locked, the entire sample chamber is pressurized and a short pressure boost is applied to push the sample to the nozzle. After the boost, the sample line is pinched to prevent sample ﬂow until acquisition or sorting is selected in the software.

Fig. 6. Loading stage in the run position.Fig. 5. Loading stage in the wash position.

The washing station is located in the back of the loading stage and is not accessible to users. When the sample input station is accessible, the washing station is automatically engaged. The washing station is used during startup, shutdown, and in between samples. This will help to reduce carryover since the sample line is cleared and the outside of the line is washed. The loading stage is locked in position while washing and sampling.

6 | S3 and S3e Cell Sorters

2.2 Fluidics System

During washing, a new sample can be loaded into the sample input station. The status of the loading stage is displayed by a locked or unlocked padlock symbol located on the touch locking system screen (Figure 7).

Fig. 7. Touch locking system screen.

To move the loading stage from the run position to the wash position:

1. Press the touch locking system screen until the padlock displays as unlocked. This will depressurize and unlock the sample station.

2. Push the loading stage handle down, pull forward, and lift up.

WARNING! The loading stage uses a spring to push up into the run or wash position. To prevent the loading stage from over-springing into position and possibly damaging the instrument, guide the loading stage into the run or wash position by holding onto the handle for a smooth movement into position.

Note: If the loading stage is in the wash position and another wash is required, press the touch locking system screen to perform washing again.

The temperature of the sample input station and the sort tube holder can be controlled from the ProSort Software via a single Peltier solid state system. The temperature range can be set from 4–37ºC in 1ºC intervals.

The sample input station is capable of mixing the sample. Sample mixing is performed by vortexing. Users can set the vortexing speed to high, low, or off, depending on the sample type.

Note: It is recommended that resuspended adherent samples are mixed at the high speed to prevent clumping.

IMPORTANT! It is highly recommended that samples are ﬁltered prior to running. This will minimize clogs in the sample tube and nozzle tip.

Instruction Manual | 7

Hardware Description

2.2.3 Nozzle

The nozzle controls many crucial aspects associated with sorting such as:

n

Creating a stable vibration to generate droplets

n

Hydrodynamically focusing the sample

n

Removing air from the nozzle (debubbling)

The nozzle compartment (Figure 8) can be accessed through the top, front sliding door. The nozzle tip is a 100 µm oriﬁce for centering the sheath stream to the laser interrogation point. The tip may be removed for cleaning or to remove a clog from the nozzle stage.

Note: If the nozzle door is opened, lasers will be shuttered, sample will be stopped, and the stream will be disabled.

IMPORTANT! Refer to Chapter 10, Maintenance, for a detailed procedure on removal, cleaning, and replacement of the nozzle assembly and nozzle tip. This is performed using the Swap Nozzle Tip wizard.

CAUTION! Shock hazard! The nozzle door is interlocked and will disable stream charge when opened. Only qualiﬁed and trained personnel can override this interlock.

A B

Fig. 8. Nozzle compartment containing the nozzle stage. A, Agilis System; B, AutoGimbal System.

2.2.4 Sort Collection Chamber

The deﬂection plates are located in the sort collection chamber (Figure 9). When these plates are electrostatically charged they direct the sorted droplets into the appropriate tubes. These plates should remain clean and dry for optimal deﬂection.

IMPORTANT! Both plates should be cleaned on a regular basis. If a signiﬁcant amount of sheath ﬂuid builds up on the plates due to a clog or misalignment, arcing between the plates can occur. When arcing is sensed by the system’s electronics, the plates will be disabled and a message will appear in the software. Using a cotton swab, clean the plates and remove any stray ﬂuid before attempting to sort.

Sorted samples can be collected into three collection vessels listed below.

n

1.5 ml tubes

n

5 ml tubes, 12 x 75 mm

8 | S3 and S3e Cell Sorters

8-well strips

Microscope glass slides

Deﬂection plates

Fig. 9. Sort collection chamber and deﬂection plates.

2.3 Optics

The sort positions for each collection vessel are numbered and will correlate to the position numbering in the software when the sort logic and limits are set. When placing tubes in the sort chamber for sorting, it is recommended to add media or buffer to the tube to help prevent sorted cells from drying out and to cushion their collection. The minimum recommended volume is 0.5 ml of media or buffer for each 5 ml tube. Quick-attaching adaptors are available for the 8-well strips or microscope slides. Adaptors are part of the accessory kit and they can be kept inside the ﬂuidics door for storage.

2.3 Optics

The S3 and S3e optics include the laser(s), mirrors, ﬁlters, and lenses, which guide the laser light to the stream of sample and collect scattered and emitted light for detection.

CAUTION! Biohazard! When running samples and sorting, hazardous aerosols may be created depending on the sample type. To prevent hazardous aerosols from spreading, keep the green containment door closed as often as possible. Consult with your local safety ofﬁcer or review local, state, and federal regulations to ensure proper handling and disposal of biohazardous substances including samples, sorted fractions, and waste.

CAUTION! Shock hazard! The sort deﬂection plates in the sort chamber are charged while sorting. An interlock on the sort chamber door will disable the plates when opened. Only qualiﬁed personnel should override this interlock.

CAUTION! Laser hazard! Laser radiation can be hazardous. Please do not override optical interlocks or remove light shields, as they are in place for your safety. Only trained personnel should access the exposed laser beam.

2.3.1 Laser

The S3 and S3e Cell Sorters are installed with a 488 nm, 100 mW laser for cell and particle interrogation that passes through the system in the sample core. The laser power and shutter can be controlled through the software interface. An optional 561 nm or 640 nm 100 mW laser are available as a second excitation source.

Instruction Manual | 9

Hardware Description

2.3.2 Beam Shaping Optic (BSO)

The beam shaping optic sits in between the laser(s) and the interrogation point. This serves to shape and focus the laser beam(s) to optimize the illumination of the cell.

2.3.3 Interrogation

The interrogation point is the point at which the laser beam(s) intercepts the core stream of the sample. At this point, light is scattered around each individual particle and the particle ﬂuoresces if any ﬂuorophores are attached.

2.3.4 Light Collection

Light is collected from two directions, forward and side angle. The light can be categorized into two types — scattered and ﬂuorescent. Scattered light refers to the wavelength of light coming from the laser, which is scattered when a particle is encountered. Fluorescent light refers to the light emitted by the ﬂuorophores or dyes attached to the particle or cell after excitation from the laser. This emitted light is of a higher wavelength than the excitation light and therefore can be separated and detected using optical ﬁlters.

2.3.5 Forward Scatter

Light coming around the particle in the forward direction (same plane as the laser beam) is collected to give an indication of particle size. By default, the scattered laser light is collected, but ﬂuorescent light could be collected by changing the ﬁlter.

2.3.6 Optical Filters

Optical ﬁlters are coated pieces of glass used to divide the spectrum of light into bands for analysis. By separating and detecting different bands of light, it is possible to understand multiple properties of each particle.

IMPORTANT! When removing or replacing ﬁlters, wear gloves to avoid depositing smudges and ﬁngerprints on the glass surfaces. Please see Section 10.5, Optical Filter Cleaning, for speciﬁc instructions on cleaning optical ﬁlters.

2.3.7 Photomultiplier Tubes (PMTs)

The PMTs are used to detect and amplify the light signals coming from each particle. They are located behind the optical ﬁlters to detect speciﬁc bands of light based on the ﬂuorophores that are attached to the cell.

2.3.8 Cameras

The S3 and S3e Cell Sorters contain several cameras for system alignment and calibration. The function of each of these cameras is detailed below.

Note: Cameras do not require adjustments by users.

n

Pinhole camera — aligns the stream and laser to the optical detection path. This camera is viewable only by service personnel and during the nozzle tip swap process

n

Droplet camera — calibrates the droplets and maintains the drop delay. The image of the break-off can be visualized in the software through the Droplet Monitor option on the home tab

n

Streams camera — calibrates the side streams and aligns them with the collection tubes. This camera can be visualized in the software through the View Streams option on the home tab

10 | S3 and S3e Cell Sor ters

2.4 Electronics

The S3 and S3e electronics process and deliver the emitted light signals to the software for user analysis. Also included in this subsystem are the deﬂection plates in the sort chamber and the safety interlocks.

CAUTION! Shock hazard! Only qualiﬁed, trained technicians should carry out service work on electronic components due to potential shock hazard.

2.4.1 Interlocks

To prevent exposure to laser light and shock hazards, the S3 and S3e systems are equipped with safety interlocks (Figure 10). On the instrument control panel of the software, the instrument status box will show visual indicators if these interlocks have been disengaged.

Note: When opened, the nozzle door will close the laser shutters to prevent laser light exposure. This interlock will also turn off the drop drive and the stream.

IMPORTANT! Open this door only if required to clear a clog or change the nozzle tip. The QC procedure must be repeated to readjust the alignment and reset the drop delay if this door is opened.

2.4 Electronics

The sort door interlock will disable the plate voltage. In addition, if sample is running it will be stopped.

Fig. 10. S3 and S3e safety interlocks.

2.4.2 Pre-Ampliﬁer

The pre-ampliﬁers are used to boost the signal coming from the PMT.

Laser shutter interlock

Sort door interlock

2.4.3 ADCs

Analog to digital converters (ADCs) convert the electrical signal coming from the pre-ampliﬁer into a digital signal and transfer that signal to the software for visualization of the data.

Instruction Manual | 11

12 | S3 and S3e Cell Sorters

ProSort™ Software

This chapter describes the features of the ProSort Software. The software is the main interface for the S3™ or S3e™ Cell Sorter System, giving overall status and providing control.

To start the software, double click the ProSort icon (Figure 11) on the desktop.

Fig. 11. ProSort Sof tware icon.

The login window will appear (Figure 12). This window will also appear when switching users. Enter your user name and password to log in. Session notes may be added and will be logged with the user session in the user report for administrative use. These notes will appear in the user report when viewed.

Note: If an automatic startup has been scheduled, the login and startup windows will appear different. Please refer to Chapter 9, Automatic Startup for details.

Fig. 12. User login window.

ProSort offers two modes of user control, administrator and user. The administrator mode provides additional control and access over user mode. Differences between the two modes are shown in Table 7. For more information on administrator privileges, refer to Section 3.3, Administrator Tab Toolbar.

Instruction Manual | 13

ProSort Software

Table 7. Control differences between administration and user modes.

Control Administrator User

 Startup • •  Shutdown • •  RunQC • •  PrintQCreports • •  PrintQCtrendingreports • •  EditQCcriteria •  Viewdroplets • •  Viewdropletsettings • •  Editdropletsettings •  Viewstreamscamera • •  Editstreamsettings •  Acquisition • •  Sort • •  Printanalysisreports • •  Printsortreports • •  Printuserreports •  Changeuserpassword • •  Changeuserrights •  Deleteusers •  Createusers •  Editusers •  Resetotheruserpasswords •

3.1 Main Software Window

The main software window contains tools for system operation and data analysis. Depending on user control mode, the main software window will either display more or fewer features.

Features available in the main software window for user mode are shown in Figure 13.

Title bar File menu and tabs

Instrument control

PMT control

Instrument status box

Workspace

Fig. 13. Main software window for user mode.

14 | S3 and S3e Cell Sorters

Status bar

3.1.1 Title Bar Buttons

The title bar of the main software window provides the items listed in Table 8.

Table 8. Title bar buttons in the main software window.

Button Command Function

Save Saves FCS ﬁle after acquiring sample

Sign out/ Signs out the current user and returns the software to the login window. If Switch user the shutdown procedure was not performed prior to sign out, the system will

Undo Certain commands can be undone and reverted to the previously existing

3.1.2 File Menu

The dropdown ﬁle menu provides the items listed in Table 9.

Table 9. File menu buttons and their function.

Button Command Function

3.1 Main Software Window

maintain startup and QC. The next user will need to log in to start a new run

state. Examples of functions that can be undone are: deleted region, deleted gate, deleted histogram or density plot, moved region or gate, creation of a plot or region, compensation adjustment

New protocol Opens a new protocol tab in the workspace

Open protocol Opens an existing protocol. An FCS ﬁle may also be selected at this time. The protocol used to acquire the data will be loaded from the FCS ﬁle

Load instrument Opens an existing protocol’s settings and loads them into the current protocol. settings The current protocol must be saved

Save protocol Saves the current protocol

Open FCS with Opens FCS ﬁle and will load data into the embedded protocol. All contents of protocol the protocol (such as plots, regions, sort logic, notes) will be displayed

Save FCS ﬁle Saves the current FCS ﬁle

Save partial Saves the last user deﬁned number of events from the acquired sample. The FCS ﬁle number of events to be saved is entered by the user before saving the ﬁle

Print Opens the print window.

Note: Use the print preview option to see where page breaks occur

Page orientation Changes between a printable landscape or portrait workspace

Preview Opens the print preview window

number, serial number, etc.)

Recent Protocols

protocols for the

Recent F CS File s

ﬁles for the user

About Displays software information (software, hardware, ﬁrmware version

About...

List of recent Lists the recent protocols used by the logged-in user

user that is logged in.

List of recent FCS Lists the recent FCS ﬁles used by the logged-in user

that is logged in.

Instruction Manual | 15

ProSort Software

3.1.3 Home Tab Toolbar

The Home tab toolbar consist of six sections for basic workspace functions and sort options (Figure 14):

n

Plots — available plots for viewing data. Table 10 describes the plots and their functions

n

Regions — different shapes for selecting region of interest. Table 11 describes the regions and their functions

n

Compensation — perform ﬂuorescence compensation. Table 12 describes the compensation buttons and their functions

n

Sort Setup — collection vessel and sort logic criteria for a run. Table 13 describes the sort setup buttons and their functions

n

Sort Monitor — monitor the sort of a run. Table 14 describes the sort monitor buttons and their functions

n

Help — opens the user manual in PDF format

Fig. 14. Home tab toolbar.

Table 10. Plot buttons and their functions.

Button Name Function

Histogram Adds a new histogram to the current protocol. The histogram can be resized

by dragging the bottom right corner

Density Adds a new density plot to the current protocol. The density plot can be

resized by dragging the bottom right corner

Sort Adds a sort plot to the current protocol. The sor t plot can be resized by

dragging the bottom corner. The sort plot graphically shows the sorts and aborts in each sort position.

Table 11. Region buttons and their functions.

Button Name Function

Rectangle Adds a rectangular region to the selected density plot. If a density plot is not

selected, this region will be grayed out. The region can be moved, resized, rotated, and/or deleted

Ellipse Adds an ellipse region to the selected density plot. If a density plot is not

selected, this region will be grayed out. The region can be moved, resized, rotated, and/or deleted

Polygon Adds a polygon region to the selected density plot. If a density plot is not

Select each desired point and move the mouse cursor outside the density plot

16 | S3 and S3e Cell Sorters

selected, this region will be grayed out

to complete. The region can be moved, resized, and/or deleted

continues

3.1 Main Software Window

Table 11. Region buttons and their functions (continued).

Button Name Function

Quadrant Adds a quadrant region to the selected density plot. If a density plot is not selected,

this region will be grayed out. The region can be moved, resized, and/or deleted

Bar Adds a bar region to the selected histogram. If a histogram is not selected, this region

will be grayed out. The region can be moved, resized, and/or deleted

Table 12. Compensation buttons and their functions.

Button Name Function

Auto Opens the Automatic Compensation Calculation Wizard window. This wizard will Compensation assist in establishing the proper compensation matrix coefﬁcients. Single control

FCS ﬁles must be acquired and saved prior to using this feature.

View Matrix Opens the Compensation Matrix window. Users can view and edit the compensation

matrix in this window

Table 13. Sor t Setup buttons and their functions.

Button Command Function

Sort Logic Opens the Sort Logic window. By default, 5 ml tube is selected for the Sort Logic

window. Users can click on the Sort Logic dropdown arrow to select 1.5 ml tube, 8-well strip, or glass slide

5 ml tube Indicates that 5 ml tube is selected as the collection vessel. Individual mode and

limits may be set for each 5 ml tube in each direction

1.5 ml tube Indicates that 1.5 ml tube is selected as the collection vessel. Individual mode and

limits may be set for each 1.5 ml tube in each direction

8-well strip Indicates that 8-well strip is selected as the collection vessel. Sort limits or mode can

be set for each well in an 8-well strip

Glass slide Indicates that glass slide is selected as the collection vessel. Sort limits or mode can

be set for each microscope slide. A maximum of 500 events can be sorted onto each location on the slide

Table 14. Sort Monitor and help buttons and their functions.

Button Name Function

Droplet Monitor Opens the Droplet Monitor window. Droplets will be visible in this window after

performing a QC procedure. The status of droplet maintenance is shown in this window. The currently set drop delay and drop drive amplitude are also shown in this window

View Streams Opens the View Streams window. This camera shows the sort streams at a point just

above the collection vessel

Sor t Statistics Opens the Sort Statistics window. The window displays sort count, sort rate,

percentage abort count, rate and %, efﬁciency, etc. Sort Statistics can be added to the print window for reporting. This window will open automatically when a sort is started

Help Opens a PDF ﬁle of the user manual as a reference

Instruction Manual | 17

ProSort Software

3.1.4 Setup and Maintenance Tab Toolbar

The Setup and Maintenance tab toolbar consists of ﬁve sections (Figure 15):

n

System — startup or shutdown the instrument from the software. Table 15 describes the system buttons and their functions

n

Quality Control (QC) — perform or review QC runs. Table 16 describes the QC buttons and their functions

n

Fluidics — access common ﬂuidic functions. Table 17 describes the buttons and their functions

n

Other — additional features. Table 18 describes the additional features and their functions

n

Publish — features for supporting publications. Table 19 describes the publish features and their functions as well as the toolbar’s remaining user buttons and their functions

Fig. 15. Setup and maintenance tab toolbar.

Table 15. System buttons and their functions.

Button Name Function

Start-Up Starts up the system by starting the sheath ﬂow, turning on the laser(s), and

backﬂushing the sample line

Use this button to start up the system if auto startup has not been selected

Shutdown Shuts down the system. The shutdown button is not active unless the loading

stage is in the wash position. The shutdown button will prompt to clean system and schedule an auto startup

Table 16. Quality control buttons and their functions.

Button Name Function

Run QC The daily QC procedure ensures optimal system performance and should

be completed every day prior to running samples. The procedure consists of placing a tube of ProLine break-off, setting up side streams, adjusting event rate, aligning nozzle, adjusting PMTs, checking the coefﬁcient of variation (CVs) and voltages, calculating drop delay, and placing the system in droplet monitor mode. The progress of this procedure will be displayed on screen

Trending Daily QC data are compiled into trending reports. These reports show trending of

CVs, PMT voltages, drop delays, etc. over a range of dates. This date range may be selected in the report screen. These reports may be saved and/or printed

QC Report This button shows and allows saving and/or printing of the daily QC report.

This report includes date, user, CV/PMT/droplet info, etc

™

calibration beads on the system, setting the droplet

18 | S3 and S3e Cell Sorters

3.1 Main Software Window

Table 17. Fluidics buttons and their functions.

Button Name Function

Debubble Removes bubbles from nozzle

Unclog Allows user to pull vacuum in the case of a nozzle clog. Use this option before

removing the tip for sonication or replacing with a new nozzle tip

Swap Tip Stops the sheath ﬂow to allow nozzle tip replacement. A wizard will appear to

walk the user through a nozzle change. Once the tip has been replaced, QC needs to be rerun to verify alignment and set the drop delay

Swap Fluidics Allows ﬂuidic containers to be swapped without shutting the system down.

Prepare a full replacement or empty container prior to pressing this button, as a three-minute timer starts after the button is pressed

Clean Cleaning can be done at low or high pressure. High pressure cleaning will cause

the system to stop maintaining droplets and a QC procedure must be run before sorting. If cleaning between samples, the low pressure option is recommended

Table 18. Other buttons and their functions.

Button Name Function

Annotation Adds an annotation window to the workspace. Annotations added prior to

saving will be saved to the FCS ﬁle’s embedded protocol

Image Allows an image to be added to the workspace. The image can be moved

or deleted. If added prior to saving, this image will be saved to the FCS ﬁle’s embedded protocol

Basic Adds basic information to the print page in the form of a header. This information Information includes date, time, user login, serial number, ﬁlename, event source. If added

prior to saving, this information will be saved to the FCS ﬁle’s embedded protocol

Filter Conﬁguration Adds the optical layout to the print page showing PMT and optical ﬁlter setup.

This can be modiﬁed to match the actual ﬁlters if they have been changed. If added prior to saving, this will be saved to the FCS ﬁle’s embedded protocol

Table 19. Publish and user buttons and their functions.

Button Name Function

Preview Displays a preview of the page layout(s) setup prior to printing

Page Orientation Changes between a printable landscape or portrait workspace

Print Opens the print dialog to select printer, number of copies, etc. and print. Note: Use the print preview option to see where page breaks occur

Print Options Adds additional windows or instrument settings to the page layout for printing.

Change Password Allows the currently logged-in user to change password

System Log Opens window for system log. Displays date, time, error, error details. This

option is helpful when working with technical support and service engineers for troubleshooting

Instruction Manual | 19

ProSort Software

3.2 Control Panel

System operation is performed and monitored with the control panel (Figure 16). The control panel consists of three parts:

n

Instrument control — controls for performing a run

n

PMT control — controls for modifying the PMTs

n

Instrument status box — displays the status of your instrument

Instrument control buttons are described in Table 20 and PMT control buttons are described in Table 21.

Sample agitation high/low button Sample station light on/off button Sample collection area light on/off button Temperature control on/off button Temperature control option

Biosafety System serial number Biosafety System ﬁrmware version Biosafety System status Time remaining on ﬁlter life Temperature inside the system

Start/Stop acquisition button Start/Stop sort button Refresh data button Pause/Restart sample acquisition

button

Eject sample tube button

Auto save sample upon reaching limit Preset sample event rate button Enter sample event rate Event limit option Cycle mode option Gate limit option

Time remaining with ﬂuidic volume Fluidics Status window button

Laser on/off button Laser power options

Fig. 16. Overview of instrument and PMT control panel.

20 | S3 and S3e Cell Sorters

Trigger channel Threshold value Alter PMT voltages Assign values for detection channels

Instrument status box Indicator of actions required or needing attention

3.2 Control Panel

Table 20. Instrument controls and their functions.

Button Name Function

Start Sample Starts sample acquisition. If a data ﬁle is loaded and it has been saved, Acquisition the data will clear and acquisition will start

Stop Sample Stops acquisition and sample ﬂow. Regions, gates, and annotations may Acquisition be modiﬁed before saving the FCS ﬁle

Start Sorting Starts sorting. If a data ﬁle is loaded and it has been saved, the data will

clear and sorting will start

Stop Sorting Stops sorting and sample ﬂow. Regions, gates, and annotations may be

modiﬁed before saving the FCS ﬁle

Clear Acquired Data Clears acquired data. All plots and statistics will be refreshed.

Pause Sample Pauses sample acquisition and sample ﬂow. The data at this point may be

appended (by the Resume function) or saved as is. Saving after pausing will disable the Resume function. To remove sample tube, unlock the touchscreen locking system located above the loading stage after pausing acquisition

Resume Sample Resumes acquisition when a sample has been paused. Resuming will

append the paused FCS ﬁle and the time paused can be seen on a time histogram. If you have removed the sample tube it must be returned and relocked before acquisition can be resumed

Eject Sample Tube Depressurizes and unlocks the sample. The loading stage can be moved

into wash position to access sample. The touchscreen locking system can also be used to unlock the sample

Adjust Sample Starts or stops agitation of the sample. There are three levels of agitation: Agitation low, high, and off. Click this button once to set to low, twice to set to high,

and three times to turn off agitation

Auto Save When checked, automatically prompted to save a data ﬁle after

acquisition stops.

Target Event Rate Allows selection of target event rate for sample acquisition or use of preset sample rates low/med/high based on sample concentration

Limit When checked, stops data acquisition after the number of events entered

has been reached

Gate Limit When checked, data acquisition stops after the number of events entered

has been reached within the selected gate

Cycle Mode When checked, automatically refreshes the data displayed according to

the entered time. The acquired data set continues to accumulate in the ﬁle

Sample Chamber Turns the sample chamber light on and off Light

Enable/Disable Sort Illuminates entire sort collection chamber for easy viewing of deﬂection Chamber Light plates, sort stream, and collection vessels

Enable/Disable Enables or disables temperature control of the sample station and sort Temperature Control collection vessel

Biosafet y Indicates the status of the biosafety system and gives information on ﬁlter life. System Status If the biosafety system loses connection, cannot connect, or if the ﬁlter life is

at 0% an error will occur and not allow the user to sort or acquire.

continues

Instruction Manual | 21

ProSort Software

Table 20. Instrument controls and their functions (continued).

Button Name Function

Fluidics System Indicates the status of the ﬂuidics system and run time information. When Status the ﬂuidics system is down to an hour of run time, the ﬂuidics status

window will automatically open to show which container needs attention. If the run time reaches 5 min, the ﬂuidics will automatically shut down

Toggle 488 nm Controls the 488 nm laser shutter. The shutter is interlocked for safety. When Laser Shutter this button is grayed out, either the laser is off or the interlock is open

Toggle 561 or 640 nm Controls the 561 or 640 nm laser shutter. The shutter is interlocked for safety. Laser Shutter When this button is grayed out, either the laser is off or the interlock is open

Adjust Laser The dropdown arrows next to each laser adjust the laser power. Actual mWatt Power power is shown by hovering over the 488 nm, 561 nm, and 640 nm labels

Table 21. PMT controls and their functions.

Button Name Function

Editable The name for each parameter is editable and will be saved with the parameter acquired FCS data. The edited parameter name will be applied to all name plot axes, compensation controls, and ﬁlter conﬁgurations

PMT Voltage Adjusts the PMT voltage for the given detector

Select trigger The trigger parameter is used to alert the system to the presence of parameter an event over the threshold. The default for this is Forward Scatter

Adjust threshold Threshold is a percentage of the trigger signal. The default value is 1.0% setting

3.3 Administrator Tab Toolbar

The administrator tab is available only for administrator users. To change a user’s privileges, refer to Section 3.3.5, Managing Users.

The toolbar for the administrator tab (Figure 17) consists of:

n

System and User Control — edit system controls and manage users

Table 22 describes the buttons in this section.

IMPORTANT! Adjusting any of these settings will impact the overall performance of the system. Only knowledgeable users should make any changes to these settings. All changes made here will apply globally to the system.

Fig. 17. Administrator tab toolbar.

22 | S3 and S3e Cell Sorters

3.3 Administrator Tab Toolbar

Table 22. Administrator tab toolbar buttons and their functions.

Button Name Function

Edit QC Criteria View and edit the CV and PMT voltage criteria for the quality control

procedure. This may be done as a result of a bead lot change

Edit Droplets Edit the settings for droplet creation. These settings include drop drive amplitude,

drop drive frequency, charge phase, defanning, and drop delay. These settings are automatically adjusted as part of the QC procedure and will result in sort changes. These settings are grayed out while the system is maintaining

Edit Streams View sort streams and edit the settings for stream creation. These settings Settings include deﬂection, charge plate voltage, defanning, phase, and test pattern

adjustment

Print User Report View usage reports including times, dates, and duration of system use for

all users

Manage Users Create new users, reset passwords for existing users, set rights, and

delete users

Decontaminate Run a decontamination wizard that will walk user through a complete ﬂuidic

system decontamination. This will take approximately 2–3 hr

Calibrate Sample Reset the differential offset sample pressures, which returns the preset Pressure Offset buttons (low, med, high) to their correct ranges

Global preferences Change global instrument options for sheath concentration used, idle

3.3.1 Editing QC Criteria

An administrator can edit the QC criteria used to pass or fail the instrument during the daily QC. This should be performed only by an experienced user who has veriﬁed system performance. The rewind arrow on the middle bottom of Edit QC Criteria window will reset the QC criteria to the factory defaults for the system (Figure 18).

When receiving a new lot of ProLine calibration beads:

1. Download the bead lot ﬁle from the S3 or S3e Cell Sorter website, www.bio-rad.com/cellsorter.

2. Click the green plus button (Figure 18).

3. Navigate to the new bead lot ﬁle.

4. Select the ﬁle and click Open.

The software will automatically update the QC criteria for the new lot of calibration beads. Values in red indicate values that are not used for pass/fail criteria for QC. If you plan to enter the bead lot information manually:

shutdown time limit, and boost adjustment, plot default options and ﬁle save default location

1. Verify the acceptance criteria on the certiﬁcate of analysis.

2. Compare the old lot to the new lot.

3. Enter the new lot information into the Edit QC Criteria window.

IMPORTANT! Changes to these criteria will apply globally to the system.

Instruction Manual | 23

ProSort Software

Fig. 18. Edit QC Criteria window with green plus button and rewind arrow highlighted.

3.3.2 Edit Droplets

Droplet controls are available to the administrator for troubleshooting purposes. While the system is maintaining, the drop delay, drop drive amplitude, and drop drive frequency cannot be adjusted (Figure 19). If any of these settings is adjusted, use ProLine calibration beads to recalculate the drop delay before running samples. The verify drop delay button at the bottom of this screen will sort three puddles onto a slide which can be examined under a microscope to conﬁrm drop delay. These puddles will sort 100 events at the current drop delay – 1, the current drop delay, and the current drop delay + 1.

CAUTION! Optimal settings are calculated automatically after performing a quality control procedure. Adjusting any of these values will change the settings and will affect the drop delay, including the break-off point.

3.3.3 Edit Streams

Streams controls are available to the administrator for troubleshooting purposes. While the system is maintaining, these settings can be adjusted. Side streams can also be turned on or off by checking the Enable Test Pattern box (Figure 20).

CAUTION! Optimal side stream settings are calculated automatically after performing a quality control procedure. Adjusting any of these values will change the settings and will affect the side streams and sort.

24 | S3 and S3e Cell Sorters

Calibrate droplets Recalculate drop delay Verify drop delay Close window

3.3 Administrator Tab Toolbar

Fig. 19. Droplet Controls window with drop settings highlighted.

Fig. 20. Streams Control window with Enable Test Pattern highlighted.

Instruction Manual | 25

ProSort Software

To check for stream defanning:

1. Locate the center stream. The stream is visualized as a small bright spot that is illuminated by the stream laser.

2. Check the Enable Test Pattern check box the system will charge the side streams to simulate a sort.

Fanning of the streams can be seen more easily with the center stream than the two side

streams. The test pattern will make the center stream slightly broader.

3. Determine if the center stream is one solid stream.

If the center stream spreads into several streams, the fanning should be adjusted.

4. Use the defanning slider until the center stream is as focused as possible (Figure 21).

5. Click the checkmark when completed to save the settings.

Fig. 21. Examples of a good center stream, A and a defanned center stream, B.

3.3.4 User Reports

User reports can be viewed, saved, printed, and exported for analysis of system usage and for billing purposes. To display a user’s usage information, select the date range of interest and click Update (Figure 22).

26 | S3 and S3e Cell Sorters

Fig. 22. User Report window with date range highlighted.

3.3.5 Managing Users

The Manage Users button opens the Manage Users window. This window allows an administrator to add, delete, or edit users (Figure 23). Passwords can be reset and users can be notiﬁed to reset their passwords from this window.

3.3 Administrator Tab Toolbar

Fig. 23. Manage Users window.

To edit a user’s information:

1. Click the pencil icon next to the user’s name.

2. Modify user information in the Edit User window (Figure 24).

3. Click Save.

Note: In the event that all administrator passwords are lost or forgotten, call your local technical support team to receive a temporary password.

Fig. 24. Edit User window.

Instruction Manual | 27

ProSort Software

3.3.6 Decontaminate

An administrator can run the decontaminate procedure if the system shows signs of heavy background, noticeable debris when running water, or contamination. The complete internal ﬂuidic system will be decontaminated. Follow the wizard procedure located in Section 10.7, Decontamination.

3.3.7 Calibrate Sample Pressure Offset

If the system shows an event rate of 0 when running calibration beads in the low setting, or if it shows 500+ events, the offset will need to be reset. Using this tool will reset the differential offset sample pressure, which will return the preset sample ﬂow rates (low/med/high) back to their correct ranges.

1. Load calibration beads into the sample station and move loading stage to the run position.

2. Click Calibrate Sample Pressure Offset.

A warning window will appear asking if the user wants to continue (Figure 25). ProLine calibration beads must be running for this calibration process.

The system will automatically begin the calibration process (Figure 26). Once complete the system offset pressure has been calibrated.

Fig. 25. Sample Calibration Warning dialog box.

Fig. 26. Calibration Sample Offset window.

28 | S3 and S3e Cell Sorters

3.3.8 Global Preferences

Users with administrator privileges have the ability to change global settings for the instrument. Changes to global preferences will become effective upon accepting the changes. Preferences speciﬁc to plots will be displayed when new plots are created.

CAUTION! These settings are global and may inﬂuence other users’ experiments. Adjusting any of these values will change the optimal settings for the instrument.

3.3 Administrator Tab Toolbar

Sheath can be used at 8x or at 1x concentration

Option to enable mandatory cleaning upon shutdown or logout

Parameter names can be preset to have user-deﬁned names displayed

Deﬁne idle shutdown option and countdown time

Choose to prompt

user to save unsaved FCS and protocols

Deﬁne default

save ﬁle locations

Fig. 27. Global Preferences window.

Boost duration can be increased or decreased

Plot default options:

Define which plot

statistics to be displayed and number of digits after the decimal

Choose which

axis parameter will be displayed and log verses linear axis values

Define plot

display size, maintain aspect ratio of plots

Display and

highlight events that are off axis

Display

compensation sliders on plots

Deﬁne emergency contact information

Sheath Fluid Options

The system can use 8x or 1x sheath solution. The default concentration is 8x. The 8x sheath ﬂuid will mix automatically with DI water prior to running through the system. One container of 8x sheath ﬂuid will provide up to 50 hours of run time compared to 9 hours of run time for one container of 1x sheath solution.

If a 1x sheath solution will be used, select 1x in the sheath concentration section prior to running the system to avoid diluting the solution.

Idle Shutdown Time

The system has the ability to shut down automatically and start the shutdown procedure after sitting idle for a designated time period. The system has a default setting of 2 hours.

This option helps to preserve laser lifetime and prevent the system from running overnight.

Instruction Manual | 29

ProSort Software

Boost Adjustment

The default boost duration is optimized to bring the sample into position for interrogation by the laser once acquisition is started. Default values can be restored by selecting Default. If this value is adjusted, test the boost duration with a known sample to optimize the duration before running the unknown samples.

Force Cleaning

There are two methods to end a user session in the software — log out or shut down. For both methods, a Force Cleaning option is available to promote cleaning and maintenance of the system. Force cleaning can help to avoid unnecessary clogs that can happen when the system is not cleaned regularly or left overnight with sample in the lines.

Default Plot Statistics

Each plot can be displayed with statistics for all events or regions drawn on the plot. Each statistic can be displayed or can be hidden by setting the preference. In addition, the number of signiﬁcant ﬁgures for each statistic can be deﬁned. Once this parameter is changed the subsequent new plots will reﬂect the new statistics defaults.

Plot Defaults

In this section there are several options that can be chosen.

n

Histogram and Density Plot axis parameters can be deﬁned and set in the global preferences. The user can choose the default axis parameter type: Area, Height, or Width. In addition, the axis can be displayed in log or linear scales. Log can be selected as the default by checking the box. Once these parameters are changed, subsequent new plots will reﬂect the new parameter defaults

n

The width and height of the plot can be deﬁned speciﬁcally. The default number of pixels is 400 x 400. In addition, the aspect ratio can be maintained if the box is checked for this option. This option applies to both histograms and density plots. Once this parameter is changed, subsequent new plots will reﬂect the new size default

n

Events may fall off scale and be displayed on or below the axis. A preference can be chosen to show an arrow indicator when events are off scale. This will help to raise awareness that some settings may need to be adjusted

n

Auto compensation is the recommended method to compensate samples properly and in a statistically correct method. Compensation sliders can be selected to be displayed or hidden on the density plot axes when the compensate box is checked. Compensation bars can also be used to make compensation adjustments, however proper gating and match medians must be used to compensate correctly

30 | S3 and S3e Cell Sorters

3.3 Administrator Tab Toolbar

Parameter Names

Each parameter is defaulted to its channel name (FSC, SSC, FL1, FL2, FL3, and FL4). The default parameter name can be entered by the user. This will be the parameter name for all new protocols created once the preferences have been changed.

Note: Parameter names can also be changed on the instrument panel but they will revert to default names when a new protocol is opened. Refer to Section 5.1.6, PMT Voltages, to see how to change names on the instrument panel.

Default Save Location

The software can autosave after each ﬁle has ﬁnished acquiring. Files can be saved to a default destination folder on the computer. This can be set for both protocols and for FCS ﬁles. In addition, there is an option to require a prompt prior to clearing data.

Emergency Contact Information

An emergency contact name and phone number can be entered into these ﬁelds. The information will be displayed in the File > About window. This will let all users of the instrument know what person within the lab or institution to contact for any emergency help with the S3 or S3e System.

Fig. 28. About window displaying emergency contact information.

Instruction Manual | 31

32 | S3 and S3e Cell Sorters

Getting Started

After system installation by a Bio-Rad certiﬁed service engineer, the S3™ or S3e™ System should not be turned off via the main power switch. The system is safe in standby mode after performing a software shutdown of the system. This will help facilitate quick or automated startup.

If the system is shut off via the main power switch, always power the instrument on before starting the ProSort running in order to communicate with the software.

IMPORTANT! Powering the system on or off incorrectly can cause the instrument to not run properly.

™

Software. The internal computer system of the instrument must be

4.1 Checking Bulk Fluidics

Prior to starting the ProSort Software, open the bulk ﬂuidics door and check the volume of each container. Ensure that the waste container is empty, and the sheath ﬂuid and DI containers are full.

Note: To change the bulk ﬂuidics after starting the software or during a run, refer to Section 5.3, Checking or Swapping Fluidics.

To reﬁll or change a container (Figure 29):

1. Detach the quick disconnect from the cap assembly by pushing in the metal button and lifting up the quick disconnect until it engages the magnetic holder.

2. Slightly lift and pull out the container.

3. Remove the cap assembly from the container.

4. Reﬁll or change the container.

5. Carefully place the cap assembly onto the container and tighten the cap.

6. Place the container into position.

7. Attach the quick disconnect to the cap assembly. An audible click should be heard.

Instruction Manual | 33

Getting Started

Sheath ﬂuid cap assembly

Fig. 29. Bulk ﬂuidics with sheath ﬂuid components noted.

Sheath ﬂuid container

Quick disconnect

IMPORTANT!

n

When handling sheath ﬂuid and DI water containers, minimize air exposure to help avoid contamination

n

While transferring cap assemblies to a new container, avoid touching the assembly to the outside surfaces of the containers. If it is necessary to place the cap assembly down, only use pre-sterilized surfaces

n

Treat all waste as bio-hazardous for safety

4.2 Logging In

To use the S3 or S3e system, ProSort Software is required. The software is preinstalled on the computer that came with the instrument.

1. Double click the ProSort Software icon located on the desktop.

2. Log into the ProSort Software.

Fig. 30. Login window.

Note: If the system has been scheduled for an automatic startup, a startup counter will be visible in the Login window (Figure 31). Please refer to Chapter 9, Automatic Startup, for startup options.

34 | S3 and S3e Cell Sorters

Fig. 31. Automatic startup message in login window.

4.3 Daily Startup

On a daily basis, the system requires approximately 20 min of warmup time before running quality control or any samples. To warm up the system:

1. Move the loading stage into the wash position.

2. Click on the Setup and Maintenance tab.

3. Click Startup.

Automatically the Startup Settings window will pop up (Figure 32). Using the startup button, the instrument can either be started up at the moment or a scheduled startup can be designated. Startup Now is the default option.

4.3 Daily Startup

Fig. 32. Startup Settings window.

4. Choose startup option in the Startup Settings window.

5. Click the checkmark box to proceed.

Note: If scheduling an auto startup, the software will return to the main window and will not start up the system until the scheduled time.

The Startup Status window will display the status of the startup process (Figure 33). The startup process includes initializing the system, ﬁlling the ﬂuidics lines, and warming up the lasers.

Fig. 33. Startup Process windows.

Instruction Manual | 35

Getting Started

During the startup process, the fundamental alignment window will also open to initiate cameras, maintain droplets, and align streams to the lasers. The startup process also includes a warm up period (Figure 34).

Fig. 34. Startup Process window.

After startup completion, both windows will close automatically and buttons in the toolbars will become available to use.

Note: Automated startup is an option for this instrument. At shutdown, a startup time can be designated. If this option is selected, be sure to empty the waste container and ﬁll the sheath ﬂuid and DI containers at the end of the day.

Note: To change the ﬂuidic containers after the software has been initiated or during a run, refer to Section 5.3, Checking or Swapping Fluidics, for instructions on performing a hot swap.

4.4 Quality Control

The quality control (QC) procedure must be run every day to ensure optimal system performance. This procedure requires the ProLine™ calibration beads to adjust alignment, verify system against the QC criteria, set up droplet creation, adjust side streams, calculate drop delay, and maintain the droplet break-off.

Note: ProLine Universal calibration beads must be used on a system with a 488/640 nm lasers. These beads can also be used on the 488 nm or 488/561 nm systems.

To perform the quality control procedure:

1. Vortex a bottle of ProLine calibration beads.

2. Place at least 10 drops (approximately 0.5 ml) into a 5 ml tube. Do not dilute these beads.

3. Place the tube with beads into the sample input station.

4. Move the loading stage into the run position.

5. Click Run QC on the Setup and Maintenance tab.

6. Choose the correct bead lot from the dropdown menu in the Event Based Alignment Settings window (Figure 35).

Fig. 35. Event Based Alignment Settings window.

36 | S3 and S3e Cell Sorters

4.4 Quality Control

Note: If the bead lot is not available in the dropdown menu, refer to Section 3.3.1, Editing QC Criteria.

A series of windows will open during the QC procedure to perform alignment and calculate drop delay settings. User interaction is not required for these windows. These steps are described later in this section.

7. After completion, the Drop Delay Alignment Status window will open with the optimal drop delay settings (Figure 36A).

Fig. 36. A , Drop Delay Alignment Status window and B, Daily QC Report.

8. Click the checkmark to open the QC Report.

9. Review the QC report (Figure 36B).

n

If the QC status passed, exit the window. The system is now ready for sample acquisition

and sorting.

n

If the QC status failed, rerun the QC procedure up to two more times.

4.4.1 Droplet Setup

The ﬁrst step in the QC process is droplet setup. Droplet setup is performed automatically and the droplets will be optimized to a stable break-off. The droplet camera image may be viewed by clicking Droplet Monitor on the Home toolbar. The Droplet Monitor window will display the drop delay, drop drive amplitude, and status of the drop delay (Figure 37).

Users with administrator privileges may access and modify these settings in the administrator tab. For more information, refer to Section 3.3, Administrator Tab Toolbar.

Instruction Manual | 37

Getting Started

Fig. 37. Droplet Monitor window.

4.4.2 Side Streams

Following droplet setup, the side streams are adjusted to align with the sort tube positions and to ensure they are not fanning. The streams may be viewed by clicking View Streams on the Home tab. The View Streams window displays a charge phase and defanning slider bar (Figure 38). Users can manually adjust the charge phase and defanning values by moving the slider bar. The Enable Test Pattern button should be selected in order to apply a charge to the stream.

Users with administrator privileges may access and modify these settings in the Administrator tab. For more information, refer to Section 3.3, Administrator Tab Toolbar.

CAUTION! Optimal side stream settings are calculated automatically after performing a quality control procedure. Adjusting any of these values will change the optimal settings and will affect the side streams and sort.

38 | S3 and S3e Cell Sorters

Fig. 38. View Streams window with charge phase and defanning slider bars highlighted.

4.4.3 Alignment

During the alignment phase, automated micro-motors sweep the ﬂuidic stream through the laser beams to ﬁnd the point of optimal illumination. This automated process requires the stream and laser(s) to be running. After alignment, the CVs and voltages are compared to the QC criteria. During the QC procedure, a calibration protocol will open and run automatically. Upon completion, the data are saved as an FCS ﬁle. This ﬁle may be opened and viewed by opening the daily QC report, selecting the desired date and time, and clicking the open FCS ﬁle button at the bottom of the report.

4.4 Quality Control

Fig. 39. QC histograms displayed in the workspace.

4.4.4 Drop Delay Determination

After the droplet and side stream formations have been optimized and the system is optically aligned, drop delay will be determined using ProDrop™ technology and set using the same ProLine Calibration Beads used for the alignment.

ProDrop technology accurately calculates the drop delay on the S3 and S3e Systems by analyzing the waste stream while running calibration particles. Events are detected normally at the nozzle and then again in the waste stream below. The drop delay setting is adjusted automatically through a set range, ﬁrst coarsely then ﬁnely, while sorting is enabled. The correct drop delay value is determined when the calibration particles are deﬂected from the center stream and therefore no longer present in the waste stream. This technology allows accurate measurement of the drop delay value without requiring the user to count beads under a microscope.

While the ProDrop technology is running, the Drop Delay Alignment Status window will open. The yellow line indicates the best drop delay during the process and this value will be set as the drop delay once completed. The green points, light green for coarse adjustments and dark green for ﬁne adjustments, indicate the number of beads detected in the waste stream. After completion, three buttons will appear in the lower right corner. These buttons, from left to right, recalculate drop delay, run a verify drop delay test, or conﬁrm and close the window (Figure 40).

Instruction Manual | 39

Getting Started

Fig. 40. Drop Delay Alignment Status window.

Recalculate drop delay Verify manually Accept

4.4.5 Drop Delay Maintenance

After drop delay determination, the system will be automatically placed into a droplet monitor mode to maintain the break-off during use. The changes required for this are completed automatically. To verify that the drop delay is being maintained, use the Droplet Monitor button on the tool bar. This window will note if the system is maintaining (Figure 41).

Fig. 41. Droplet Monitor window with status and recalculate button highlighted.

40 | S3 and S3e Cell Sorters

4.4.6 Recalculate Drop Delay

To recalculate the drop delay using ProLine calibration beads, click the Recalculate button (Figures 40 and 41). This button will not align the streams or verify CVs. This button will only calculate the drop delay.

During the drop delay calculation in the QC procedure, the same recalculate button on the Drop Delay Alignment Status window will be available in the Drop Monitor window. During the QC procedure, recalculation can be performed until values are acceptable, prior to clicking the checkmark box.

4.4.7 QC Criteria

Data collected during the QC procedure are checked against the QC criteria after the system has been aligned. Users with administrator privileges may access and modify these settings in the Administrator tab. For more information, refer to Section 3.3, Administrator Tab Toolbar.

CAUTION! Adjusting any of these values will affect the overall system performance.

For each ﬂuorescent parameter, two criteria may be set:

n

PMT voltage — the PMT voltage required to bring the population to channel 128 must be below the set point for each parameter

n

Coefﬁcient of variation (CV) — the CV is measured in each parameter and must be under the set point

4.4 Quality Control

4.4.8 QC Report

A QC Report window will open after completing the alignment and drop delay settings. This window displays the QC status and whether the system has passed or failed the QC requirements. This report can be saved or printed (Figure 42).

To view a previous QC Report, on the Setup and Maintenance tab select QC Report > Date.

IMPORTANT! These values may need to be changed to account for lot-to-lot variation of the ProLine calibration beads.

Print report Open FCS ﬁle Save data ﬁle

Fig. 42. QC Report window with QC status highlighted.

Instruction Manual | 41

Getting Started

4.5 Protocols and Workspace

Before acquiring samples, a protocol with at least one histogram or density plot is required to visualize the events. The last used protocol will automatically open with the software. A protocol consists of histograms, gates, regions, trigger, threshold, PMT settings, sort logic, compensation settings, and all other information required for a run. Protocols can be used as a template for future runs.

To add a new protocol:

1. Click File.

2. Select New Protocol from the dropdown menu. A new protocol will open as a new tab in the workspace.

3. From the Home tab, add a histogram or density plot to your workspace. Plots will automatically open in the workspace in the order they were added.

4. Click on a plot to activate the window. A blue frame will appear indicating items within the window can be changed.

5. Change the parameters; resize window; add regions, bars or quadrants; apply gates; or show statistics within this window.

6. Repeat for other plots.

To close a protocol, use the X symbol alongside the Protocol tab (Figure 43).

Fig. 43. Creating new protocols.

A protocol can be saved using the File dropdown menu. Protocols that have not been saved are marked with a dot in the title. After a protocol is prepared, samples can be acquired using the protocol or an existing FCS ﬁle can be embedded into the current protocol. Refer to Section 4.5.4, Opening FCS Files, to view existing data ﬁles.

4.5.1 Opening Protocols

Protocols can be opened from the File menu (Figure 44). When selected, the Open Protocol option will bring up a Windows prompt to locate and open the desired protocol. To the right of the ﬁle name ﬁeld is a dropdown box for protocol or FCS ﬁle. By selecting the FCS ﬁle option, a protocol can be opened directly from a saved FCS ﬁle.

42 | S3 and S3e Cell Sorters

4.5 Protocols and Workspace

Fig. 44. File dropdown menu with New, Open, Load Instrument Settings, and Save Protocol highlighted.

4.5.2 Loading Instrument Settings

Protocols can be saved for future use. Each protocol is saved with the following information: plots, regions, gates, trigger, threshold, PMT voltages, sort logic, sort limits, sort modes, compensation settings, and ﬁlter conﬁguration.

To load a previously saved instrument setting:

1. Open the protocol to be used or create a new protocol and save.

2. Click File > Load Instrument Settings.

3. Choose the protocol that contains the settings to be loaded.

The preexisting settings will be loaded into and displayed in the current protocol and will be ready to use for acquisition or sorting.

4.5.3 Saving Protocols

To save a protocol (Figure 44), click File > Save Protocol.

Refer to Section 3.3.8, Global Preferences, for information on how to assign default folders for saving both protocols and FCS ﬁles.

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Getting Started

4.5.4 Displaying Multiple Protocols

Multiple protocols can be opened at the same time by opening an existing protocol or a new protocol from the ﬁle menu. When multiple protocols are opened, they will be displayed as tabs along the top of the workspace (Figure 45). Selecting between each tab will update instrument settings with the information contained in each protocol.

Fig. 45. Workspace with protocol tab highlighted.

4.5.5 Opening FCS Files

The ProSort Software allows an FCS ﬁle to open into two different types of protocols (Figure 46). The data can be embedded into the currently opened protocol by using the Open FCS Data Only option. The FCS ﬁle can also be opened into the original protocol in which it was acquired by using the Open FCS with Protocol option. The existing gates and plots associated with that protocol will be recalled to the workspace.

44 | S3 and S3e Cell Sorters

Fig. 46. Opening and saving FCS ﬁles.

4.5.6 Saving FCS Files

When the AutoSave box is not checked, the FCS ﬁle can be saved after acquisition or sort. The protocol used during the acquisition or sort is embedded in the FCS ﬁle. The ﬁle can be opened at a later time for analysis or to be used as a template for a subsequent acquisition.

4.5.7 Saving Partial FCS Files

There may be instances when a large number of events will be acquired during a sort. These FCS ﬁles can become very large and potentially can cause the software to run more slowly when reopening for analysis. In addition, it may be difﬁcult to view populations with large numbers of events displayed at one time. It is possible to save a partial FCS ﬁle during a sort by saving only a set number of events (Figure 47). Click File > Save Partial FCS File. A window will appear to select how many events to save. The selected number of events will be saved from the end of the sort. Sort statistics from the entire sort will be saved regardless of the partial of the FCS data saved.

For example, if 500,000 events are sorted and 20,000 events are saved as a partial FCS ﬁle, then the last 20,000 events sorted (480,001–500,000) will be saved to the ﬁle.

4.5 Protocols and Workspace

Fig. 47. Save Partial FCS File Events window.

4.5.8 Histograms and Density Plots

Histograms and density plots can be created from the Home tab. Clicking on each button will create the plot and add it to the protocol workspace. Each plot will be added to the right of the prior plot and will wrap to the next row as needed. Plots will be created with the default parameters of FSC for histograms, and FSC and SSC for density plots. Refer to Section 3.3.8, Global Preferences, for information on how to change plot defaults for axis, log vs. linear, plot size, and plot display options.

4.5.9 Resizing, Moving, and Deleting Plots

When plots are added to the workspace they are tiled automatically based on the size of the screen.

To resize histograms or density plots:

1. Hover over the desired plot.

2. Hold down the handle in the lower right corner and drag the plot to the desired size.

Instruction Manual | 45

Getting Started

Plots can be moved within the workspace by grabbing the bar at the top and dragging to the desired position. The remaining plots will shift accordingly. To delete a plot, click the red X in the upper right corner. This will delete the selected plot (Figure 48).

Fig. 48. Density plot with resizing, moving, and deleting functions highlighted.

4.5.10 Changing Parameters

To change the parameters displayed in a plot, click the dropdown arrow and select a parameter (Figure 49). If a log or compensated parameter is desired, check the appropriate box.

Fig. 49. Density plot with parameters displayed.

46 | S3 and S3e Cell Sorters

4.5.11 Viewing Statistics

To view the statistics on a plot, highlight the plot and select the Statistics dropdown arrow in the lower left corner (Figure 50). To hide the statistics, click the same button.

4.5 Protocols and Workspace

Fig. 50. Density plot with statistics.

4.5.12 Editing and Reordering Statistics

There are several options for displaying statistics. Changes in the settings will be saved with the protocol (Figure 50). Refer to Section 3.3.8, Global Preferences, for information on how to change the default statistics.

To remove statistic headings:

1. Right click in the Statistics panel.

2. Check or uncheck statistics to display in the statistics table.

3. Check Apply to all to apply to all plots in the protocol.

To reorder statistic headings:

1. Click the statistic name in the Statistics title bar.

2. Drag and drop to the desired location.

Instruction Manual | 47

Getting Started

1. Right click on the Statistics panel.

2. Click on the statistic name and drag and drop to the new location.

3. Check Apply to all to apply to all plots in the protocol.

To alter number of digits after the decimal:

1. Right click in the Statistics panel.

2. Find statistic of interest and change the value in the Precision box.

3. Check Apply to all to apply to all plots in the protocol.

4.5.13 Creating Regions

Regions can be created in a histogram or density plot to further identify populations of interest. A bar region is the only option on a histogram and all other regions will be unavailable. Density plots may have ellipses, rectangles, quadrant, or polygon regions. There are two ways to create a region on the plot — by using the Home tool bar or by right clicking on the graph.

To create a region using the Home toolbar:

1. Click on the desired plot.

2. Select the region from the Home toolbar.

To create a region using the right click on the mouse:

1. Click on the desired plot.

2. Right click within the plot and choose a region type (Figure 51).

The selected region will appear in the desired plot. Region buttons will be grayed out if the incorrect plot is selected.

Fig. 51. Right-click option for creating regions.

48 | S3 and S3e Cell Sorters

4.5 Protocols and Workspace

A region can be moved, tilted, resized, and deleted (Figure 52).

1. Click the desired region.

2. To move region, hover till the crosshairs icon appears. Click and drag to new location.

3. To tilt, click the green circle indicator above the region and drag to change the angle.

4. To resize, click the circle or square indicators around the region and drag to the appropriate size.

5. To delete, right click within the region and choose Remove region.

Fig. 52. Moving or tilting a region within a plot.

4.5.14 Creating Gates

Gates can be used between plots once regions have been created. The purpose of gating is to limit the events shown in one plot by a region in another plot.

To create a gate (Figure 53):

1. Select the plot to be gated.

2. Right click within the plot.

3. Select Apply Gate to the region or regions of interest.

The regions on the gated plot will appear in the title bar of the plot. To ungate a plot, right click in the gated plot and select Clear Gate.

Fig. 53. Density plot with Apply Gate selected.

Instruction Manual | 49

50 | S3 and S3e Cell Sorters

Acquisition

Sample acquisition can be done independent of sorting. As a sample is run through the system, data are gathered and can then be saved in FCS 3.1 format. These ﬁles can be loaded and analyzed using third party software compatible with FCS 3.1 ﬁles. Often, setup samples — for example, unstained samples or single-color compensation samples — are acquired prior to sorting the actual sample of interest.

5.1 Acquisition Setup

Prior to running a sample, a protocol must be loaded or created. At least one histogram or density plot is required to begin acquisition. Protocols can be created new or loaded using a protocol ﬁle or from a previously acquired FCS ﬁle. Refer to Section 4.5, Protocols and Workspace, for information on how to create or open protocols. Access all instrument controls via the ProSort™ instrument control panel.

To set up an acquisition or sort:

1. Create or open a protocol.

Note: At least one histogram or dot plot must be created to continue.

2. Load the sample onto the sample input position and move the loading stage into the run position.

3. Detectors can be renamed for each parameter based on ﬂuorophores or markers used.

4. Check cycle mode to allow refresh of the data displayed.

5. Deﬁne target event rate, trigger parameter, and threshold value.

6. From the instrument control panel, click the Start Sample Acquisition button.

7. Adjust PMT voltages to place populations.

8. Adjust target event rate, the trigger parameter, and threshold value.

9. Stop sample acquisition by pressing the same button.

10. Check and deﬁne event limit. Gate Limit can be chosen. Cycle mode can be unchecked at

this time.

Note: Data can be displayed in hyperlog if the compensation box is checked.

Optional: AutoSave can be enabled to automatically prompt to save ﬁle after event limit is reached.

11. Re-collect sample and save FCS ﬁle if AutoSave is not enabled.

12. Unlock and remove sample tube. Move the loading stage into the wash position before the next sample is run.

Instruction Manual | 51

Acquisition

Note: Cycle mode can be enabled or the refresh button can be used to refresh the data displayed on the screen as adjustments are being made.

When running multicolored experiments, compensation is necessary. In order to use the Auto Compensation wizard, single stained compensation control samples must be saved in addition to the multicolored stained samples.

13. Run single stained compensation control for ﬁrst parameter.

14. Set event limit high enough to visualize positive population; collect and save ﬁle.

15. Repeat and collect samples for each parameter.

16. Use the Auto Compensation wizard to set compensation for multicolored samples.

Refer to Section 5.2.1, Auto Compensation Wizard, for instructions on compensation methods.

Depending on the ﬂuorophores used in the experiment, ﬁlters may need to be optimized in order to capture signal properly. Please refer to Section 5.4, Optical Filters, for information on how to create custom ﬁlter conﬁgurations.

Refer to Section 3.2, Control Panel, for an overview of the instrument control functions.

5.1.1 Target Event Rate

The event rate is related to the sample concentration. Generally, for every 1,000,000 cells per ml sample concentration, the event rate may be increased by 1,000 events per second. As an example, if the sample is concentrated to 10,000,000 cells per ml, the maximum event rate should be 10,000 events per second.

The target event rate is dependent on the trigger and threshold settings. A target event rate can be entered into the event rate box in the instrument control panel (Figure 54). The software will maintain the entered rate throughout the acquisition or sort if the sample concentration is appropriate for the target event rate. The actual target event rate is shown to the right of the target event rate and on the status bar at the bottom of the software screen. The maximum target event rate while acquiring is 100,000 events per second.

The button for preset sample event rate can also be used. The sample pressure is preset for low/med/high rates. The sample rate will be dependent upon the concentration of the sample.

Fig. 54. Target event rate.

52 | S3 and S3e Cell Sorters

5.1.2 Event Limit

An event limit may be set on the acquisition (Figure 55). This limit will be based on the total events collected. The gate default is All. When this event limit is reached, the acquisition will stop and the FCS ﬁle can be saved. The maximum number of events acquired in a single FCS ﬁle is 100,000,000.

Note: Large FCS ﬁles may take time to save, load, and display.

Fig. 55. Event limit and cycle mode.

A gate limit can be used to deﬁne the event limit. Each gate created on the plots will be listed on the dropdown menu. Once a gate is chosen, the acquisition will continue until the event limit is reached within the gate.

5.1 Aquisition Setup

5.1.3 Cycle Mode

At the beginning of an acquisition, it may be helpful to utilize the cycle mode for a quicker refresh of the data displayed while changing the settings (Figure 55). Cycle mode is set in units of seconds and will progressively display events that fall within that period of time. Older data will be not be displayed.

Use this function when adjusting PMT voltages and/or threshold and trigger settings. After data and populations of interest appear where desired on your plots, disable this mode and restart acquisition for normal cumulative acquisition.

5.1.4 Trigger

The trigger parameter is the initial parameter of detection to signal the system that a particle of interest is present. Combined with the threshold setting, the trigger parameter deﬁnes real events which should be detected and analyzed (Figure 56). Typically, a scatter parameter is selected for the trigger, as it will identify all particles above a given size regardless of the ﬂuorescent signal, though any parameter can be used for this function.

Fig. 56. Target event rate and threshold level.

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Acquisition

5.1.5 Threshold

The threshold level is set using the trigger parameter with a range of 0.1–100% (Figure 56). The purpose of the threshold is to set an intensity level to eliminate any background noise and debris while still allowing real events to be detected and analyzed.

5.1.6 PMT Voltages

Each parameter is collected by a photo multiplier tube (PMT). Independent voltage control is available for each PMT. These voltages can be adjusted to bring the populations of interest on the histogram or density plot scale (Figure 57). Custom names for each parameter can be entered by clicking on the parameter of interest on the PMT control panel. Changing this name will update the names on all plot axes, in the ﬁlter conﬁguration window, and on the compensation matrix. Refer to Section 3.3.8, Global Preferences, for information on how to set default parameter names.

Fig. 57. PMT control panel with name changed on the FL1 parameter.

5.1.7 Auto Save

An auto save option is available as a check box on the instrument control panel. If this box is checked (Figure 58), a prompt will appear to name and save the FCS ﬁle after an acquisition or sort is completed.

Fig. 58. Checked auto save box.

54 | S3 and S3e Cell Sorters

5.1.8 Pausing and Resuming

Acquisition may be paused and resumed while in progress using buttons on the instrument control panel. Pausing will stop the sample ﬂow. During the pause, the time parameter within the data ﬁle will continue, and will show a time gap when resumed.

If you wish to replace the sample tube during the pause:

1. Press the touch locking system screen above the loading stage until the padlock is displayed as unlocked.

2. Move the loading stage into the wash position.

3. Replace the sample.

4. Move the loading stage into the run position.

To resume, click the Resume button on the instrument control panel.

IMPORTANT! If the loading stage is not placed in the wash position after a sample is removed and before a new one is loaded, the system will not provide a pressure boost. Without washing the sample line, contamination in the new sample may occur.

5.1.9 Stopping and Saving

When the desired data have been collected, acquisition can be stopped and the data saved. The data are saved in FCS 3.1 format and will include the protocol used for acquisition. The stop button will depressurize and unlock the sample. Before running another sample, place the loading stage in the wash position to backﬂush and clean the sample line.

5.2 Compensation

5.1.10 Backﬂushing and Washing

After the sample is unlocked, the loading stage can be moved to the wash position. The wash process includes a backﬂush and an exterior wash of the sample line. This process is crucial to reducing carryover and for overall cleanliness of the system. After washing, the loading stage will automatically unlock, and the next sample may be placed into the run position.

If an additional wash is desired, you have two options:

n

Lower the loading stage for >2 sec then bring it back to the wash position. An additional wash cycle will run after returning to the wash position

n

Press the touch locking system screen above the loading stage until the padlock displays as locked. This will initiate an additional wash

5.2 Compensation

Compensation is the process by which spectral overlap (spillover) is removed from other detectors. The inherent overlap of emission spectra from ﬂuorescent labels and probes makes compensation necessary. This is of particular importance when simultaneously measuring ﬂuorescence from several labeled antibodies or probes. During acquisition, compensation can be adjusted to eliminate this spectral overlap between ﬂuorophores (Figure 59).

The density plot shown displays an FL1 control data ﬁle (Figure 59). The FL1 positive population is higher on the FL2 scale than the negative population, though it has no FL2 ﬂuorescence. When compensation has been applied, the positive FL1 population has the same median value as the FL1 negative population in the FL2 parameter.

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Acquisition

Fig. 59. Density plot showing uncompensated (left) and compensated data (right).

The same can be done for the FL2 positive control, though the compensation required is much less (Figure 60).

Fig. 60. Same data plots showing incorrectly compensated ﬁle (left) and correctly compensated ﬁle (right).

As compensation is adjusted, the compensation matrix will be updated. At any time the compensation matrix can be viewed using the button on the toolbar (Figure 61).

56 | S3 and S3e Cell Sorters

Fig. 61. Compensation matrix.

5.2.1 Auto Compensation Wizard

The compensation wizard assists with establishing the proper compensation matrix coefﬁcient for each parameter. Single controls for each parameter and the associated FCS ﬁles must be saved prior to compensation.

5.2 Compensation

To use the compensation wizard:

1. Run single controls for each parameter and your multi-stained sample.

2. Save FCS ﬁles.

3. Select Home > Compensation Wizard.

4. Select the parameters for compensation (Figure 62).

5. Click the ﬁle icon for each selected parameter (Figure 62).

Fig. 62. Use the auto compensation wizard to select individual parameter FCS ﬁles and adjust regions.

Instruction Manual | 57

Acquisition

6. Locate and select the single control FCS ﬁle for each parameter.

7. Select whether to use height or area for all parameters.

8. Click the forward blue arrow to open the Automatic Compensation Calculation Wizard window (Figure 63).

9. Adjust the scatter, negative, and positive regions as necessary.

Note: The positive and negative population gates must not overlap.

Fig. 63. Using the auto compensation wizard to adjust positive and negative gates.

10. Click the blue forward arrow to go to the next control.

11. Repeat steps 9 and 10 for remaining controls.

12. After all controls are adjusted, click the checkmark button. This will complete the automatic compensation calculation.

13. Check the compensation box on the axis of the desired histogram or density plot to display the compensated data and acquire or sort.

14. The compensation matrix may be viewed at this point if desired.

The hyperlog feature allows data that have values <0 to be correctly displayed. This data display format is used only for compensated data (Figure 64). It converts a small portion of the scale around zero to linear, and adds a small amount of negative log scaling to each access, preventing negative events from piling up on the axis. This allows better visualization of compensated data.

Note: The area parameter is grayed out and cannot be changed because compensate is checked. Compensation can only be applied to area or height depending on which selection was chosen during auto compensation.

58 | S3 and S3e Cell Sorters

Fig. 64. Density plot showing hyperlog, log, and compensation options.

5.2 Compensation

5.2.2 Manual Compensation

It is advised to use the auto compensation feature of the software, however it is possible to alter the compensation matrix manually (Figure 65). This process, although historically used, is difﬁcult to set correctly and the process of compensation should only be performed using the wizard unless knowledgeable and statistically correct adjustments can be done by manual compensation. Compensation controls for each ﬂuorochrome you will be using in your experiment. The process is most accurate if you have both positive and negative events in each sample, and it is advisable to acquire at least 5,000 positive events. After the sample is acquired, load each sample one by one into the software, setting the histogram axes to the parameters you would like to adjust, and checking the Comp box on the histogram axes. By applying quadrants it is possible to attempt to match the median of the negative population to the positive population (Figure 64). When the value in the matrix is changed, the populations will move. Alter until the median values are correct and matched.

Fig. 65. Compensation matrix.

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Acquisition

5.3 Checking or Swapping Fluidics

The bulk ﬂuidic containers are located inside the system and can be accessed by the ﬂuidics door on the left front of the instrument. These ﬂuidic containers require changing on a regular basis. As recommended in Chapter 4, Getting Started, ﬂuidic containers should be changed prior to initiating the software or after shutting down the system. If the ﬂuidic containers need to be swapped after startup or during a run, the S3™ and S3e™ Cell Sorters are designed to easily allow this without the need to shut down the system. This is considered as a hot swap of the ﬂuidics.

The ﬂuidics status indicator on the instrument control panel provides information regarding the levels and approximate run times remaining for these containers (Figure 66). This window will pop up when the approximate run time is down to an hour and will continue to alert every 15 min until a swap is performed. A hot swap can be done when the limiting ﬂuid is highlighted. The alert may be snoozed at this point if desired.

WARNING! When the remaining run time is down to 5 min, the system will automatically shut down. The ﬂuidics will need to be reﬁlled and the startup procedure must be performed to ensure the lines contain no air and the system is properly QC’d.

Fig. 66. Fluidics Status window.

5.3.1 Hot Swap

A hot swap is deﬁned as the process of changing a ﬂuidic container after initial instrument startup or during a run without the need to shut down the instrument. Unlike other cell sorters, the S3 and S3e Systems maintain the ﬂuidics pressure internally to enable a hot swap for uninterrupted sort collection and eliminates the need for additional repressurization time.

IMPORTANT! New containers should be prepared before beginning a hot swap, as the swap time is limited to three min before the system will automatically shut down due to low ﬂuid levels.

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5.3 Checking or Swapping Fluidics

To perform a hot swap:

1. On the Setup and Maintenance tab select Swap Fluidics.

2. A countdown timer will automatically start. This is the amount of time left to swap the containers before the system will automatically shut down (Figure 67).

WARNING! The system contains a limited amount of reserve ﬂuids to continue running the system during the swap. If the timer reaches zero, the system will automatically shut down. Work quickly and carefully during these swapping steps. If the system shuts down, perform the QC procedure prior to running a sample.

Fig. 67. Swap Fluidics window with swap time remaining highlighted.

3. Open ﬂuidics door.

4. Detach the quick disconnect from the cap assembly by pushing the metal button.

5. Lift the quick disconnect until it engages the magnetic holder. This will hold the quick disconnect away from exchange area.

For sheath ﬂuid or DI water:

6. Slightly lift and pull out the empty container.

7. Remove the cap assembly from the empty container.

8. Uncap and remove the seal from a new 4 L container.

9. Carefully place the cap assembly onto the new 4 L container and tighten the cap.

10. Proceed to step 11.

For Waste:

6. Slightly lift and pull out the waste container.

7. Remove the waste cap assembly.

8. Empty the waste container following lab procedures for liquid biohazardous and chemical waste removal.

9. Place the waste cap assembly into a new designated waste container or recap emptied waste container.

10. Proceed to step 11.

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Acquisition

11. Place the container back into position.

12. Attach the quick disconnect to the cap assembly. An audible click should be heard.

13. Click the play button in the Swap Fluidics window to ﬁnish the swapping procedure.

IMPORTANT!

n

When handling sheath ﬂuid and DI water containers, minimizing air exposure will help to avoid contamination

n

While transferring cap assemblies to a new container, avoid touching the assembly to outside surfaces of the containers. If it is necessary to set the cap assembly down, use only sterilized surfaces

n

Treat all waste as biohazardous for safety

5.4 Optical Filters

The optical ﬁlters separate and direct the ﬂuorescent light to the PMTs for detection. These ﬁlters are located behind a sliding panel on the top and toward the rear of the S3 or S3e System.

5.4.1 Standard Filter Conﬁguration

The standard ﬁlter conﬁgurations for the S3 or S3e Systems are shown in Figure 68. The ﬁlter conﬁguration can be viewed and edited in the ProSort™ Software.

To view or edit the ﬁlter conﬁguration:

1. On the Setup and Maintenance tab select Filter Conﬁguration.

2. Click inside each area to modify the ﬁlter name or wavelength.

3. Click the checkmark button to save the changes.

WARNING! Changes to the ﬁlter conﬁguration will be set as the default.

Fig. 68. Different ﬁlter conﬁgurations. A, 488/561 nm system; B, 488/640 nm system; C, 488 nm system.

62 | S3 and S3e Cell Sorters

In front of each PMT is a holder for a neutral density ﬁlter or bandpass ﬁlter. Each ﬁlter holder contains a screw-in retaining ring that can be removed or replaced with the provided red S3 spanner wrench.

5.4.2 Optical Filter Blocks

The S3 and S3e Systems include two preset ﬁlter blocks. The ﬁlter set depends on the instrument conﬁguration. The optical ﬁlter blocks can be changed to accommodate dectection of different ﬂuorophores. These blocks can be easily removed with the silver handle (Figure 69).

5.4 Optical Filters

Fig. 69. Lifting an optical ﬁlter block.

The ﬁlter blocks contain the dichroic and bandpass ﬁlters. The optical ﬁlters within the block are user changeable and will accommodate multiple ﬁlter sizes as described in Table 23. The dichroic ﬁlter holders are locked from the bottom of the block. Once unlocked, they can be removed and replaced from the top of the block.

Table 23. Description of ﬁlter sizes for use with optical ﬁlter blocks.

Filter Descripiton

Normal incidence ﬁlters 25.4 mm (1 in) round, up to 2 mm thick

12.7 mm (0.5 in) round, up to 2 mm thick 11 x 11 mm square, up to 2 mm thick 12 x 25.4 mm rectangle, up to 2mm

Dichroic holders 25.4 mm (1 in) round, up to 2.7 mm thick

12 x 25.4 mm rectangle, up to 2.7 mm thick

Neutral Density (ND) holders 25.4 mm (1 in) round, up to 10 mm thick

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64 | S3 and S3e Cell Sorters

Sorting

Sorting cell populations is highly dependent on several factors such as cell type, population frequency in the starting sample, cell concentration, sample preparation, and sort logic deﬁnitions. It is very important to start with a single cell suspension with minimal amount of dead cells or debris contamination.

6.1 Sort Setup

The majority of sort setup procedure is automatic and is completed during daily QC. When ready to sort, the desired populations must be selected using regions and gates. At this time sort logic deﬁnitions must be set.

To start a sort:

1. Click File > New Protocol or Open Protocol.

2. Click Home > Histogram or Density to add plots to the workspace.

3. Adjust temperature control for sample input station and sort collection area if desired.

4. Place ﬁltered sample into the sample input station.

5. Move the loading stage into the run position.

6. Click Start on the instrument control panel to acquire a small amount of the sample.

7. Set sorting regions and gates on plots with populations of interest.

8. Right click on the desired region to be sorted and select sort direction.

9. Repeat with a second region if two-way sorting is desired.

10. Click the Sort Logic dropdown menu to select the collection vessel of choice.

11. Deﬁne the sort logic parameters in the Sort Logic window (Figure 70) by setting the sort mode (purity, enrich, or single) and event limit for each collection position.

12. Deﬁne sort collection tube volume if you want to track collected volume to prevent collection overﬂow.

13. Place sort collection vessel in the sort collection device at positions 1, 2, etc.

Note: 5 ml tubes should have at least 0.5 ml media and 1.5 ml tubes should have at least

0.1 ml ﬁltered media to collect and cushion the sorted cells.

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Sorting

Fig. 70. Sor t Logic window.

14. Close the sort chamber door.

15. Click Start Sort on the instrument control panel. The Sort Statistics window will open and sorting will begin (Figure 71). The Sort Statistics window can be reopened using the Sort Statistics button on the Home toolbar. Click on the arrow in the upper right corner to expand the window and show the statistics table.

16. If no limits have been set, click the same sort button to stop the sort when desired.

17. Return loading stage to the washing position.

18. Open sort chamber door and remove sorted cells.

Fig. 71. Sort Statistics window.

66 | S3 and S3e Cell Sorters

6.1.1 Sort Collection

The S3™ and S3e™ Systems offer four types of sort collection vessel options:

n

5 ml tubes

n

1.5 ml tubes

n

8-well strips

n

Microscope glass slides

Each sort direction has the capability of sorting into 5 ml tubes and 1.5 ml tubes up to a quantity of 5 tubes in each direction. Using the quick-attaching adaptors, sorted cells can be collected into 8-well strips (up to a quantity of 8) or onto a microscope slide (up to a quantity of 20). Each tube and well may have different sort modes and limits assigned.

It is highly recommended that media, buffer, or serum is added to the sort tubes or wells prior to sorting to give the cells a cushion, avoid evaporation of droplet volumes, and to help preserve cell viability during the sort collection period. The minimum recommended volume is 0.5 ml of ﬁltered buffer or media for each 5 ml tube. The minimum starting volume for 1.5 ml tube is 0.1 ml.

Note: The sort output may be temperature controlled to the same temperature as the sample input station. This temperature can be enabled/disabled and adjusted on the instrument control panel.

6.1 Sort Setup

6.1.2 Volume Tracker

During a sort, the system has the ability to track the volume and number of droplets deﬂected into each sample collection tube. Volume tracking can be enabled to automatically stop a sort or move to the next tube position before a tube overﬂows.

To enable this feature:

1. Click the Prevent Overﬂow checkbox in the Sort Logic window (Figure 72).

2. Enter the starting volumes for the sort collection tubes.

Note: The minimum starting volume for a 5 ml tube is 0.5 ml and the maximum is 2 ml. The minimum starting volume for 1.5 ml tube is 0.1 ml and the maximum is 0.5 ml.

Fig. 72. Sort Logic window with Prevent Overﬂow highlighted.

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Sorting

Note: If a sort limit is selected and volume tracking enabled, whichever criterion (limit or full tube) is met ﬁrst will stop the sort.

IMPORTANT! The maximum total volume for a 5 ml collection tube is considered to be 4 ml. The maximum total volume for 1.5 ml tube is 1 ml. Going above these maximum volumes will cause spraying and risk contamination.

6.1.3 Setting Regions to Sort

Regions and gates in the histograms and dot plots are used to create sort logic. Multiple regions can be used to establish the sort logic (Figure 73).

To set the sort logic:

1. Identify the population of interest with regions and gates.

2. Right click in the region to sort and select Right Sort or Left Sort. That region will change color and any regions used in the gating of that plot will change as well. Regions used in sort criteria may be conﬁrmed in the Sort Logic window and sort statistics panel.

Fig. 73. Choosing sort deﬁnitions.

6.1.4 Sort Logic and Collection Vessel

After regions and gates have been assigned to sort left or right, the sort collection vessel must be selected. The Sort Logic window can be used to review sort logic, set sort modes and limits, and set priority (Figure 74). The selected collection vessel will be displayed as the icon to show the sort type selected. If there is overlap between the logic of the two streams, the left sort direction will take priority unless changed. The numbers shown in this window represent the numbers in the sort output area.

68 | S3 and S3e Cell Sorters

Fig. 74. Sort Logic window with sort priority and sort modes highlighted.

6.2 Sort Modes

Table 24 describes the three sort modes available when setting up a sort. Sort modes can be selected for each sort direction.

Table 24. Sort mode deﬁnitions.

Sort Mode Deﬁnition When to Use

Single Cell This mode requires that one and only one positive event be in the Single cell deposition

center of the droplet without any negative events nearby. Recovery will be reduced due to the sort restrictions

Purity This mode will sort as long as no negative events are present in the General sorts where both purity droplet. If multiple positive events are present, sorting will still occur and recovery are important

Enrich This mode will sort all positive events regardless if negative events Sorts when recovery is critical are nearby. Purity will be reduced to maintain high recovery and purity is not as important

6.3 Sort Statistics

The sort statistics window can be viewed during a sort and added to a print page for reporting (Figure 75). This window will automatically open when a sort is started.

If a saved partial FCS ﬁle is opened, aggregate statistics can be shown:

1. File > Open FCS File with Protocol.

2. Click on the Home tab and click Sort Statistics.

3. Check the Show Aggregate Statistics box to view statistics from all sorted events.

Fig. 75. Sor t Statistics window.

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Sorting

Table 25 describes the available sort statistics. Each statistic is sort-stream speciﬁc. The Sort Statistics window can be placed in the workspace for printing. Sort statistics are saved within the FCS ﬁle.

Table 25. Description of sor t statistics.

Statistics Deﬁnition

Sor t logic The regions used to identify the populations to sort

Mode The sort mode used for sorting left or right

Current target The sort target position used during the sort

Sort count The current number of positive events sorted

Sor t volume (ml) The volume sorted into each tube/well. This does not include the media placed

in the tube prior to sorting

Abort count The current number of positive events aborted

Sor ts per second The sort count per second as a rate

Aborts per second The abort count per second as a rate

Sort % Number of sorted events divided by total events x 100

Abort % Number of aborted events divided by total events x 100

Efﬁciency Number of sorted events divided by the sum of sorted and aborted events x 100

Event rate Real-time events per second during the sort

Total events Total events acquired during the sort

Sor t time Sort time elapsed

Additional information can be shown in the Sort Statistics window by using the arrow on the upper right hand side of the window. This will expand the window to show a table listing the data for each tube during the run. There are up to ten outputs equivalent to ﬁve tubes in two directions. This information can be saved as a tab-delimited ﬁle using the save button located in the lower right corner of the window. This information is automatically stored in the FCS ﬁle.

Fig. 76. Sort Statistics window expanded to reveal additional information.

70 | S3 and S3e Cell Sorters

6.4 Sort Plots

During a sorting experiment a sort plot can be created in the current protocol. This plot displays graphically the counts of events (sorts and aborts) per tube/well/slide spot in the sort run. The data on this sort plot (Figure 77) correspond to the Sort Statistics window (Figure 76). The numbers 1–10 are the sort position designations as assigned in the Sort Logic window (Figure 74). Regions can be applied to the sort plot. These regions can be applied to other histograms or density plots.

6.3 Sort Statistics

Fig. 77. Sort Plot window.

Instruction Manual | 71

72 | S3 and S3e Cell Sorters

Additional Software Features

The ProSort™ software is an acquisition and sorting software package with the additional features described in this chapter.

7.1 Debubble

During the startup procedure, a debubble step will occur to clear the system of air. At times, a bubble may enter the nozzle and cause problems with the stream. On the Setup and Maintenance tab select Debubble to rinse the system and attempt to debubble the nozzle. The stream can also be monitored at this time to see any changes.

7. 2 Un c log

A clog can easily be introduced and can cause multiple problems with the ﬂuidics. When a clog occurs, the software will display an error, stop the sample, stop the sort, and eject sorted samples for protection.

Use the Unclog button on the Setup and Maintenance tab toolbar as a ﬁrst-line action. The

™

and S3e™ Systems have the ability to backﬂush the nozzle, potentially releasing the clog

S3 and ﬂushing it into the waste. If successful, run a couple of debubble cycles to remove any air that may have been pulled into the nozzle body.

7.3 Swap Tip

There will be occasions when the unclog function will not be able to clear a clog in the system. Use the Swap Tip button on the Setup and Maintenance tab toolbar to run the Swap Tip wizard. This wizard will walk through each step of the nozzle tip swap. Use the same Swap Tip button when nozzle adjustments are necessary or the nozzle needs cleaning.

Please refer to Section 10.4, Swap Nozzle Tip Wizard, for step-by-step instructions on swapping the nozzle tip, or contact Technical Support if any of these solutions do not resolve the problems.

IMPORTANT! To avoid clogs when working with cells, resuspend cells into single cell suspension and always use a 40 µm or smaller ﬁlter prior to cell sorting.

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Additional Software Features

7.4 Clean System

In between samples or users, cleaning can be done at low or high pressure. High pressure cleaning will cause the system to stop maintaining droplets and a QC procedure must be run before sorting. If cleaning between samples, the low pressure option is recommended. The ﬂuidics lines will be ﬂushed thoroughly with cleaning solution, similarly to the cleaning protocol used during the shutdown or logout procedure.

To clean the system:

1. Fill a 5 ml tube with a minimum of 3 ml of cleaning solution.

2. Load tube into the sample station and move in to run position.

3. Click the Set Up and Maintenance tab.

4. Click Clean System.

The cleaning protocol will automatically begin. A prompt will appear to warn the user that the clean function will begin and that the contents within the sample tube will be used rapidly.

Fig. 78. Cleaning protocol warning box.

5. Select the cleaning mode to run (Figure 79).

6. Click Ye s to proceed with mode (Figure 80).

7. Move the loading stage into wash position (Figure 81).

Fig. 79. Clean System modes.

Fig. 80. Warning for high pressure cleaning protocol.

74 | S3 and S3e Cell Sorters

Fig. 81. Cleaning protocol ﬁnal stage.

The cleaning protocol will be completed when the window disappears. If a high pressure cleaning has been chosen, run a QC using ProLine™ calibration beads before sorting.

7.5 Instrument Status Box

The instrument status box is located at the bottom of the instrument control panel. The notiﬁcation and the color of the box will change depending on the state of the instrument. Table 26 describes the notiﬁcations and colors.

7.5 Instrument Status Box

Table 26. Instrument Status Box notiﬁcations and colors.

Color Notiﬁcation Action Example

Gray Off Nothing Off

Green Ready Can begin acquisition or sort Acquiring sorting

Yellow Information Perform requested action Require QC before proceeding Fluids Low/High

Orange Warning Nothing, system is running Calibrating

Red Error Perform required maintenance Not maintaining droplets

7.6 Status Bar

The status bar is located at the bottom of the ProSort Software window with nine status indicators (Figure 82). Table 27 describes the status indicators.

Fig. 82. Status bar with the status indicators numbered.

Table 27. Description of each status indicator.

Number Description

1 Software connectivity to instrument

2 Current date

3 Temperature control status

4 Event rate (events per second)

5 Total event number

6 Acquisition time elapsed

7 Sort time elapsed

8 Current user

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Additional Software Features

7.7 Printing

Print and print preview buttons are available in the File dropdown menu and on the Setup and Maintenance tab (Figure 83). Use the print preview function to move histograms and density plots in your print layout. Regions can also be modiﬁed in this view and will apply to the protocol displayed. Once arranged, click the print button to bring up the print dialog where the printer and paper may be selected. Sort statistics, ﬁlter conﬁguration, annotations, and compensation matrix may also be printed on the page. Select the desired print options on the Setup and Maintenance tab.

Fig. 83. Printing options available.

7.8 Quality Control Reports

Two different reports are available to track the daily performance of the S3 and S3e Systems. One is the daily QC report which will show pass/fail and droplet information for the selected day (Figure

84). Past days can be viewed using the date select option. If QC has been run multiple times in a

given day, select the daily QC report of interest from the Time dropdown list. To open the FCS ﬁle for the QC data shown in the report, select the Open FCS File button at the bottom of the report. This report may also be printed if desired.

Fig. 84. Daily QC report.

76 | S3 and S3e Cell Sorters

7.9 User Reports

The second QC report is a trending report in which a range of dates can bev viewed for PMT voltages, CVs, and drop drive amplitude and frequency (Figure 85). When viewed, comments can be added and the reports may be printed for reference.

7.9 User Reports

User reports are accessible by administrators and can be used to track system usage over time (Figure 86). These reports can be printed for reference and will include the session notes entered when users were logged into the software.

Fig. 85. QC trending report.

Fig. 86. User report.

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Additional Software Features

7.10 Biosafety System

The S3™ Biosafety System is a Class I aerosol containment hood designed to work with the S3 and S3e Cell Sorter, which works in conjunction with ProSort™ Software, providing users and the environment protection from aerosols created during the cell sorting process.

The S3 Biosafety System is fully integrated with and monitored by the S3 and S3e Cell Sorter’s ProSort Software, so users have real-time information about HEPA ﬁlter life and the temperature of the system inside. To maintain containment, the fan speed is regulated based on the cell sorter operation mode and the airﬂow rate through the HEPA ﬁlter.

The S3 Biosafety System functionally meets airﬂow requirements from the National Institutes of Health (NIH) standards for Class I biosafety and the International Society for Advancement of Cytometry (ISAC) guidelines.

For more information please see the S3 Biosafety System Class I manual.

Fig. 87. S3 Biosafety System Class I status window.

IMPORTANT! The S3 Biosafety System must remain on at all times when the S3 or S3e Cell Sorter is inside the system. The cell sorter should be left with the main system power on and will enter into idle mode when not in use.

Should the S3 Biosafety System be turned off when the S3 or S3e Cell Sorter power is on inside, there could be risk of overheating and it may become a ﬁre hazard.

If any of the following occur, the S3 and S3e Cell Sorters will stop sample acquisition or sort and alert the user:

n

Airﬂow through HEPA ﬁlter drops below required limit

n

Filter life reaches 0%

n

ProSort Software loses communication with the enclosure

Until resolved, the software will not allow running samples or sorting.

When performing the swap tip procedure, the front panel may be lifted and attached via magnet to the upper ﬁlter housing to allow nozzle access. Note that in this state, the S3 Biosafety System’s airﬂow is reduced and the system is not considered to be in Class I containment.

IMPORTANT! If the airﬂow through the HEPA ﬁlter falls below required limits for containment, the software will alert the user and stop the sort or acquisition immediately.

78 | S3 and S3e Cell Sorters

Shutdown

8.1 Daily Shutdown

The S3™ and S3e™ System should be shut down at the end of the day. This will turn off all droplet formation and charging, lasers, and streams. A ﬂush will be completed with the DI water container to rinse the system.

IMPORTANT! Always follow the personal protective equipment (PPE) guidelines relevant to your laboratory’s safety procedures for dealing with ethanol or bleach.

Note: At any time, the shutdown procedure can be canceled by clicking the X icon on the dialog windows.

Note: If a user chooses to log off from the software, the force cleaning procedure option can be run. Similar to the shutdown procedure, windows will appear with the exception of the automatic startup window.

To shut down:

1. Ensure that the sample loading stage is in the wash position.

2. On the Setup and Maintenance tab select Shutdown. The software will conﬁrm with a dialog box that you intend to shut down the system (Figure 88).

Fig. 88. System shutdown check.

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Shutdown

3. Click Yes to conﬁrm shutdown.

Note: If there are any unsaved protocols, the software will warn you that unsaved changes will be lost (Figure 89).

4. Click Yes to continue or No to abort shutdown procedure.

Fig. 89. Unsaved protocols check.

The shutdown procedure will display an automatic startup option. For ease of use and efﬁcient workﬂow, the system can be automatically started the next day (Figure 90).

5. If using the automatic startup option, select Yes and set the restart date and time.

Fig. 90. Automatic Restart window with shutdown icon highlighted.

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8.1 Daily Shutdown

A window will open requesting a tube of cleaner to be used to clean the system (Figure 91).

6. Load a tube of cleaning solution.

7. Move the loading stage into the run position.

Fig. 91. Star t the clean system option by following the prompt to load a tube of cleaner.

8. The system will automatically run through the cleaning procedure, which takes approximately 2 min, while displaying status windows (Figure 92).

9. Put the Loading Stage into the wash position.

Fig. 92. Cleaning status windows.

Instruction Manual | 81

Shutdown

Once the system is clean, the shutdown procedure will shutdown hardware, ﬂuidics, air will be purged, rinsing and backﬂushing of the sample line, rinsing and backﬂushing the sample probe and lastly clearing the waste catcher (Figure 93).

The user will then be logged out and if automatic startup has been entered the countdown will begin.

Fig. 93. Shutdown Status windows.

IMPORTANT! It is advised to clear waste and reﬁll ﬂuidic containers at the end of the day’s use.

For automatic startup, waste must be empty and 8x sheath ﬂuid and DI water must have a supply sufﬁcient for a 2 hr run time, minimum, in order to start up the system properly without errors.

Once the shutdown procedure is completed the software will return to the opening login screen.

At this time the system is fully shut down and can be left in standby mode till the next use.

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header

Automatic Startup

The ProSort™ can schedule an automatic startup of the system. Upon automatic start up, the system will verify the stream is coarsely aligned and no clogs are present. If these conditions are not met, the stream will be shut off automatically to avoid spraying or ﬂooding. A warning will appear and the system may be manually started to resolve the issue.

9.1 Scheduling an Automatic Startup

There are three ways to schedule an automatic startup.

1. Using the Startup button (provided no scheduled startup has been set). Please refer to Section 4.3, Daily Startup.

2. During the shutdown procedure. Please refer to Section 8.1, Daily Shutdown.

3. During the decontamination procedure. Please refer to Section 10.7, Decontamination.

Fig. 94. Startup settings.

Instruction Manual | 83

Automatic Startup

Fig. 95. Automatic Restart window during the shutdown procedure.

Fig. 96. Automatic restart window during the decontamination procedure.

9.2 Previously Scheduled Automatic Startups

After double clicking the ProSort Software icon on the desktop, a login window will appear. If a previous user has scheduled an automatic startup of the system, a countdown will appear at the bottom of the login window (Figure 97). This countdown indicates how much time is left before the startup procedure will begin.

Note: The software must be opened to the login screen for the auto startup to occur.

Fig. 97. User login window with auto startup countdown.

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9.2 Previously Scheduled Automatic Startups

When a user logs onto the system and an automatic startup time is designated, the software will display the Auto Startup Scheduled window (Figure 98).

There are three options that can be performed at this time. These options determine whether the automatic startup will proceed as scheduled or be canceled.

Fig. 98. Auto Startup Scheduled window.

WARNING! Canceling a previously scheduled automatic startup may inﬂuence other users’ plans. Be sure to check with others when choosing this option.

Table 28. Auto star tup buttons.

Button Command Function

Cancel Cancels the previously scheduled automatic startup. A message will

appear in the login screen indicating that a user has cancelled the automatic starup

Accept and Logs current user out of system and continues with scheduled sign out automatic startup

Allow auto startup Keeps scheduled automatic startup time but allows the user to start

up the system and run experiments in the meantime. The user can also alter the startup time

Instruction Manual | 85

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Maintenance

Always follow the PPE guidelines relevant to your laboratory’s safety procedures for dealing with the chemicals recommended below.

10.1 General Maintenance

10.1.1 Daily

It is recommended that the sample line is cleaned every day prior to shutdown. See Section

10.6, Disinfectants, for details regarding approved cleaners for this tubing.

The system must be shut down through the software on a daily basis. If the system is not shut down properly, it may be prone to software-instrument communication problems, and the sample lines, nozzle, and ﬂuidics may be prone to contamination.

If using the automated startup option, be sure to clear the waste and ﬁll the ﬂuidics at the end of use to ensure no errors will occur.

10.1.2 Weekly

Each week, wipe down the system with a mild disinfectant. Clean any salt crystal buildup in the sort chamber or on the deﬂection plates.

Inspect the camera windows for any debris or salt. Clean them with a swab and mild disinfectant as needed.

10.1. 3 Ye a r l y

It is recommended to purchase the annual preventative maintenance (PM) plan offered with the S3™ and S3e™ Cell Sorter. The PM includes but is not limited to an annual onsite visit by a service engineer to:

Replace the inline sheath ﬁlter

Replace the inline DI ﬁlter

Replace the bulk ﬂuid ﬁlters in the

Replace the air pump ﬁlter

Replace the inline ﬁlters

sheath and DI bottles

Replace the rinse ﬁlters

Clean the optical ﬁlters

Replace the nozzle assembly

Examine tubing and connectors

Perform system check

Verify and adjust optical alignment

Instruction Manual | 87

Maintenance

10.2 Dealing with Clogs

A clogged or partially clogged nozzle tip can occur while analyzing or sorting and it is usually a result of an unﬁltered sample delivering clumps of cells to the tip. When this happens, the stream will be unable to spray straight or will be blocked completely. Several things can be done to remove this clog. These options, in order of severity are listed below. Once complete, click the startup button and allow the system to verify that the clog has been removed.

10.2.1 Unclog Option in Software

A clog can easily be introduced and can cause multiple issues with the ﬂuidics. Resuspend cells into a single cell suspension and always use a 40 µm or smaller ﬁlter prior to cell sorting to help avoid clogs. If a clog does occur, use the Unclog button on the Setup and Maintenance tab toolbar as a ﬁrst-line action. The S3 and S3e Cell Sorters have the ability to backﬂush the nozzle, potentially releasing the clog and ﬂushing it into the waste.

If successful, a couple of debubble cycles are required to remove any air that may have been pulled into the nozzle body.

If unsuccessful, see Section 10.2.2, Run Sample of Cleaner, or Section 10.4, Swap Nozzle Tip Wizard.

10.2.2 Run Sample of Cleaner

Running a sample with 70% ethanol or 10% sodium hypochlorite could help free the clog. This is run through the sample input station. Cleaning solution can dislodge or loosen clogs to free up the line or the nozzle tip.

If successful, run at least two rinse cycles prior to running any test samples.

If unsuccessful, swap nozzle tip (Section 10.4).

10.3 Cleaning or Replacing Nozzle Tip

The nozzle is the main ﬂuidics component of the system and provides stable sheath ﬂow, focused sample introduction, and droplet creation and charging. The nozzle tip is ﬁxed at a 100 µm tip oriﬁce that provides centered sheath stream for laser interrogation. The tip may be removed to clean or to remove a clog. The nozzle can be accessed through the top, front sliding cover.

Note: The cover is interlocked and lasers will be turned off if it is opened.

Note: When performing any of the following instructions, use the Swap Nozzle Tip button to

open the wizard, which will assist in swapping or cleaning the tip. The wizard will also walk through restarting and aligning the system.

88 | S3 and S3e Cell Sorters

10.4 Swap Nozzle Tip Wizard

The Swap Tip wizard will walk through each step necessary to perform a successful nozzle tip swap. Read through the wizard windows carefully and follow instructions on each screen as you proceed with the protocol.

Clear an area around the instrument to have adequate working space. At times, adjustment to the nozzle stage is necessary. Handle the nozzle with clean gloves.

First, determine which nozzle system is installed in your cell sorter (Table 29).

Table 29. Cell sorter and nozzle systems.

Catalog # Cell Sorter Nozzle System Proceed to Section

145-1001, 145-1002 S3 Cell Sorter Agilis 10.4.1

145-1001, 145-1002 S3 Cell Sorter (upgraded with AutoGimbal

145-1005, 145-1006, 145-1008 S3e Cell Sorter AutoGimbal 10.4.2

10.4.1 Nozzle Tip Wizard with the Agilis System

The Agilis system uses two axis-motorized positioners that are controlled by the software. These piezo-driven micromotors help align the stream to the pinhole and to the collinear lasers. In addition, there are three gimbaling screws that are used to make manual adjustments to the nozzle stage for nozzle height in relation to the pinhole and to the streams laser.

™

feature)

10.4 Swap Nozzle Tip Wizard

AutoGimbal 10.4.2

The nozzle tip must still be removed, cleaned, and reseated manually prior to alignment. The stream must be hitting the waste hole to ensure successful results.

To swap the nozzle tip:

1. On the Setup and Maintenance tab select Swap Tip. The Swap Nozzle Tip wizard will open (Figure 99). Follow the instructions indicated by the wizard.

2. Open the nozzle chamber door.

3. Press on the left side of the black metal light protection device and lift out of the way.

4. Locate the thumb screw hinge.

5. Loosen the thumbscrew until the nozzle body rises up and the tip can be seen.

6. Click the blue arrow.

Fig. 99. Nozzle swap wizard window.

Instruction Manual | 89

Maintenance

Fig. 100. Nozzle swap wizard window.

7. Place a lint-free cloth in the nozzle chamber under the nozzle tip to prevent dropping the nozzle into the instrument.

8. Rotate the black collar holding in the nozzle until it comes loose.

Note: There is a small O-ring that will be removed as well.

9. Pull out the small white nozzle tip.

If sonication is desired or necessary:

10. Place the tip into a tube containing DI water.

11. Cap the tube.

12. Place the tube into a sonicator for several minutes.

13. Proceed to step 14.

If sonication of the tip is not performed:

10. Using a 1 ml syringe ﬁlled with ethanol, press against the top and eject ethanol through the nozzle tip, rotating between the 100 µm tip and the larger opposite end to free clog.

11. Watch the resulting stream. The ﬂuidic stream should be coming out straight from the tip.

12. Repeat if necessary until the stream is coming out straight.

13. Proceed to step 14.

14. Place the O-ring at the point on the nozzle where the bottom of the black collar will be.

15. Place the nozzle tip back onto the nozzle assembly by pushing it up using the sides of the tip. Avoid touching the end.

16. Secure the black retaining collar until ﬁnger tight.

17. Remove the lint-free cloth from nozzle chamber.

18. Press the stage back down and tighten the thumb screw.

19. Click the blue forward arrow.

90 | S3 and S3e Cell Sorters

Bio-Rad S3 User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Introduction

1.1 System Components

1.2 Installation Requirements

Hardware Description

2.2 Fluidics System

2.3 Optics

2.4 Electronics

ProSort™ Software

3.1 Main Software Window

3.2 Control Panel

3.3 Administrator Tab Toolbar

Getting Started

4.2 Logging In

4.3 Daily Startup

4.4 Quality Control

4.5 Protocols and Workspace

Acquisition

5.2 Compensation

5.3 Checking or Swapping Fluidics

5.4 Optical Filters

Sorting

6.2 Sort Modes

6.3 Sort Statistics

6.4 Sort Plots

Additional Software Features

7.4 Clean System

7.5 Instrument Status Box

7.6 Status Bar

7.7 Printing

7.8 Quality Control Reports

7.9 User Reports

7.10 Biosafety System

Shutdown

Automatic Startup

9.2 Previously Scheduled Automatic Startups

Maintenance

10.2 Dealing with Clogs

10.3 Cleaning or Replacing Nozzle Tip

10.4 Swap Nozzle Tip Wizard