BIO RAD S3, 145-1001, 145-1002 Instruction Manual

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S3™ Cell Sorter

Instruction Manual

Catalog #145-1001

#14 5-1002

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 Telex: 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

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.

S3 Cell Sorter 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

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Safety and Regulatory Compliance

For safe operation of the S3 cell sorter system, 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 cell sorter system.

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 system waste container, adhere to the recommended precautions and guidelines in this manual, and comply with any local guidelines speciﬁc to your laboratory and location

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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 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 system conforms 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 cell sorter system is designed to operate safely when used in the manner prescribed by the manufacturer. If the S3 system 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 system should be performed only by Bio-Rad personnel.

Biohazards

The S3 cell sorter system is a laboratory product. 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

S3 Cell Sorter Instruction Manualiv |iv |

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 system includes 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 system includes a waste container that may potentially contain hazardous chemical materials depending on the sample used.

Explosive or Flammability Hazards

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

Electrical Hazards

The S3 system poses 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 system 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 system can be stored under the following conditions:

Temperature range 5–35°C

Relative humidity 20–70%

Disposal

The S3 cell sorter system contains 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 countr y-speciﬁc instructions.

Warranty

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

| vS3 Cell Sorter Instruction Manual | v

S3 Cell Sorter Instruction Manualvi |vi |

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 f t ware 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 88

10.5 Optical Filter Cleaning 94

10.6 Disinfectants 94

10.7 Decontamination 95

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 99

References 103

S3 Cell Sorter Specifications 105

Ordering Information 107

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S3 Cell Sorter Instruction Manualviii |

Introduction

The S3™ cell sorter is designed to offer affordable, dependable, and simpliﬁed cell sorting. As a benchtop cell sorter, the S3 system is 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 cell sorter creates 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 cell sorter includes a complete internalized ﬂuidics and temperature control 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.

S3 Cell Sorter Instruction Manual | 1

1.1 System Components

The S3 cell sorter complete system includes the following components:

S3 Cell Sorter instrument, 1 each

n

S3 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 system 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 cell sorter can be installed by a service engineer, a site must be 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.

27.5 x 25.5 x 25.5 in

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Hardware Description

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

2.1 System Overview

The S3 system consists of ﬂuidics, optics, electronics, and software. It 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

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2.1.1 Instrument Back Panel

The rear connector panel of the S3 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 system also includes 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 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 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

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.

S3 Cell Sorter Instruction Manual | 5

Quick disconnect

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.

6 | S3 Cell Sorter Instruction Manual6 |

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.

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.

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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.

Fig. 8. Nozzle compartment containing the nozzle stage.

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

S3 Cell Sorter Instruction Manual8 |

8-well strips

Microscope glass slides

Deﬂection plates

Fig. 9. Sort collection chamber and deﬂection plates.

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 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.

2.3.1 Laser

The S3 system is 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.

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.

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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 system contains 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 Cell Sorter Instruction Manual10 |

2.4 Electronics

The S3 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 system is 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.

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

Fig. 10. S3 safety interlocks.

2.4.2 Pre-Ampliﬁer

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

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.

Laser shutter interlock

Sort door interlock

| 11S3 Cell Sorter Instruction Manual | 11

12 | S3 Cell Sorter Instruction Manual12 |

ProSort™ Software

This chapter describes the features of the ProSort software. The software is the main interface for the S3 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.

S3 Cell Sorter Instruction Manual | 13

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.

S3 Cell Sorter Instruction Manual14 |

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

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

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

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

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.

Recent FCS Files

ﬁles for the user

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

that is logged in.

S3 Cell Sorter Instruction Manual | 15

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 sort 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

selected, this region will be grayed out

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

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

16 | S3 Cell Sorter Instruction Manual16 |

continues

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 but tons 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

Sort 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

| 17S3 Cell Sorter Instruction Manual | 17

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 Cell Sorter Instruction Manual18 |

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

| 19S3 Cell Sorter Instruction Manual | 19

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 Cell Sorter Instruction Manual20 |

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

Instrument status box Indicator of actions required or needing attention

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