BD FACSCanto Reference Manual

For In Vitro Diagnostic Use

bdbiosciences.com

Part No. 642241 Rev. A
June 2007

BD FACSCanto

Flow Cytometer

Reference Manual

BD Biosciences

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© 2007, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced,
transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any
form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior
written permission from BD Biosciences.

The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its
products and services at any time to incorporate the latest technological developments. Although this guide has been
prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions,
nor for any damages resulting from the application or use of this information. BD Biosciences welcomes customer
input on corrections and suggestions for improvement.

BD FACSDiva software © Becton, Dickinson and Company. This software is the property of Becton, Dickinson and
Company. Each sale of a stored unit of this software grants the purchaser a nontransferable, nonexclusive, personal
license. This software may not be duplicated, reproduced, or copied in any form or by any means whatsoever, except
as otherwise permitted by law.

BD FACSCanto clinical software © Becton, Dickinson and Company. This software is the property of Becton,
Dickinson and Company. Each sale of a stored unit of this software grants the purchaser a nontransferable,
nonexclusive, personal license. This software may not be duplicated, reproduced, or copied in any form or by any
means whatsoever, except as otherwise permitted by law.

BD, BD logo, and all other trademarks are property of Becton, Dickinson and Company.

Cy™ is a trademark of Amersham Biosciences Corp. Cy™ dyes are subject to proprietary rights of Amersham
Biosciences Corp. and Carnegie Mellon University and are made and sold under license from Amersham Biosciences
Corp. only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham
Biosciences Corp., 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.

JDS Uniphase is a trademark of JDS Uniphase, Inc.

Microsoft and Windows are registered trademarks of Microsoft Corporation.

Sapphire is a trademark and Coherent is a registered trademark of COHERENT, INC.

Teflon is a registered trademark of E.I. du Pont de Nemours and Company.

All other company and product names might be trademarks of the respective companies with which they are
associated.

Patents

PerCP: US 4,876,190
APC-Cy7: US 5,714,386

FCC Information

WARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance
could void the user’s authority to operate the equipment.

NOTICE: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant
to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference
when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio
frequency energy and, if not installed and used in accordance with the instruction manual, can 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 or her own expense.

Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits.

This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulations.

Cet appareil numérique de la classe A respecte toutes les exigences du Réglement sur le matériel brouilleur du
Canada.

Notice

BD Biosciences delivers software and workstations that are intended for running the cytometers supplied by
BD Biosciences. It is the responsibility of the buyer/user to ensure that all added electronic files including software
and transport media are virus free. If the workstation is used for Internet access or purposes other than those specified
by BD Biosciences, it is the buyer/user’s responsibility to install and maintain up-to-date virus protection software.
BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation.
BD Biosciences is not liable for any claims related to or resulting from buyer/user's failure to install and maintain
virus protection.

History

Revision Date Change Made

336928 Rev. A 1/04 Initial release

337969 Rev. A 4/04 Updated for CE IVD release

338619 Rev. A 9/04 Updated for BD FACSDiva software v4.1

343370 Rev. A 9/05 Updated for BD FACSCanto clinical software v2.0 and the Opticon LG2 Imager barcode reader

640801 Rev. A 5/06 Updated for BD FACSDiva software v5.0 and BD FACSCanto clinical software v2.1

642241 Rev. A 6/07 Updated for BD FACSDiva software v6.0

Contents

About This Manual ix

Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Chapter 1: Introduction 13

Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Fluidics Cart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Computer Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

BD FACS Loader (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Barcode Reader (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Chapter 2: BD FACS Loader Option 33

Loader Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Using the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Changing Operational Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Maintaining the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Cleaning External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

v

Chapter 3: Barcode Reader Option 45

Installing and Using the Barcode Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Cleaning the Barcode Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

1D Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

2D Barcode Symbologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Chapter 4: Maintenance 51

Scheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Emptying the Waste Container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Purging the Fluidics Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Decontaminating the Fluidics System (Long Clean) . . . . . . . . . . . . . . . . 59

Replacing the Air Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Replacing Fluidics Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Unscheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Changing a Cubitainer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Removing an Air Lock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Cleaning External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Removing Bubbles from the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Cleaning the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Purging the Bubble Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Decontaminating the Fluidics System for Storage . . . . . . . . . . . . . . . . . . 80

Replacing the Bal Seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Resetting the Cytometer Circuit Breaker . . . . . . . . . . . . . . . . . . . . . . . . . 84

Reconnecting the Ethernet and RS232 Cables . . . . . . . . . . . . . . . . . . . . . 85

Reconnecting the Fluidics Cart Tubing . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Replacing the Fluidics Level Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Replacing the Fluidics Cart Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

vi BD FACSCanto Flow Cytometer Reference Manual

Chapter 5: Troubleshooting 95

Cytometer Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Fluidics Cart Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Loader Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Appendix A: Technical Overview 105

Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Fluidics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Optics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Optical Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Detector Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Spillover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Electronics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Pulse Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Appendix B: Supplies and Replacement Parts 127

Cytometer Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Installation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Other Replacement Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Bar Code Reader Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Cytometer Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Labware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Contents vii

Appendix C: Technical Specifications 133

Cytometer Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Signal Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Fluidics Cart Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Loader Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Index 141

viii BD FACSCanto Flow Cytometer Reference Manual

About This Manual

This manual contains reference information about the BD FACSCanto™ flow
cytometer and BD FACS™ Loader. Operating instructions are contained in the
BD FACSCanto Instructions for Use.

Most cytometer functions are controlled by BD FACSCanto™ clinical software
and BD FACSDiva™ software. BD FACSCanto clinical software contains
modules for dedicated clinical applications with automatic gating algorithms,
while BD FACSDiva software is non–application specific. You can use either
software to perform cytometer quality control. Each software package has its
own reference manual, provided on the documentation CD.

BD Biosciences recommends that first-time users of this cytometer take
advantage of operator training offered with the sale of every new cytometer.

The BD FACSCanto Flow Cytometer Reference Manual assumes you have a
working knowledge of basic Microsoft® Windows® operation. If you are not
familiar with the Windows operating system, refer to the documentation
provided with your computer.

ix

Conventions

The following tables list conventions used in this manual. Table 1 lists symbols
that are used in this manual or on safety labels to alert you to a potential hazard.
Text and keyboard conventions are shown in Table 2.

Tabl e 1 Hazard symbols

Symbol Meaning

CAUTION: hazard or unsafe practice that could result in material damage,
data loss, minor or severe injury, or death

Electrical danger

Laser radiation

Biological risk

a. Although these symbols appear in color on the cytometer, they are in black and white throughout this

reference manual; their meaning remains unchanged.

Tabl e 2 Text and keyboard conventions

Convention Use

; Tip

NOTICE

Italics Book titles and new or unfamiliar terms

a

Features or hints that can save time or prevent difficulties

Provides information that supplements the topic material

> Menu selection

For example, “select File > Print” means select Print from the File
menu.

Ctrl-X Press the indicated keys simultaneously

For example, Ctrl-P means hold down the Control key while
pressing the letter p.

x BD FACSCanto Flow Cytometer Reference Manual

Technical Assistance

For technical questions or assistance in solving a problem:

• Read the section of the manual specific to the operation you are
performing.

• See Chapter 5, Troubleshooting.

If additional assistance is required, contact your local BD Biosciences technical
support representative or supplier.

When contacting BD Biosciences, have the following information available:

• product name, part number, and serial number

• software version number

•error messages

• details of system performance

For cytometer support within the US, call [phone].

For support within Canada, call [phone].

Customers outside the US and Canada, contact your local BD representative or
distributor.

About This Manual xi

xii BD FACSCanto Flow Cytometer Reference Manual

1

Introduction

The BD FACSCanto system uses a fixed-optics design and advanced digital
electronics to support multicolor analysis of up to six fluorescent markers and
two scatter parameters per assay.

The cytometer does not require special facilities—it plugs into a standard wall
outlet, uses air-cooled lasers, and provides its own air pressure and vacuum for
the fluidics and waste. You can prepare samples on the BD FACS™ Sample Prep
Assistant II and import the sample prep worklist.

For further automation, use BD FACSCanto clinical software and the BD FACS
Loader for sample acquisition. Alternatively, use BD FACSDiva software for
more flexibility in acquisition and analysis.

See the following for more information:

• Intended Use on page 14

• System Components on page 14

• System Requirements on page 31

13

Intended Use

The BD FACSCanto system is intended for use as an In Vitro Diagnostic device
for identification and enumeration of lymphocyte subsets in human cells in
suspension for flow cytometry.

System Components

The BD FACSCanto system consists of a benchtop flow cytometer, a self­contained fluidics cart, and the BD FACSCanto workstation.
System options include an automated sample loader and a barcode reader.

For a description of system components, see:

• Flow Cytometer on page 15

• Fluidics Cart on page 25

14 BD FACSCanto Flow Cytometer Reference Manual

• Computer Workstation on page 28

• BD FACS Loader (Optional) on page 28

• Barcode Reader (Optional) on page 30

Flow Cytometer

Cytometer power is controlled by the power button. All other cytometer and
fluidics cart functions are controlled by BD FACSCanto clinical software and
BD FACSDiva software (provided with the cytometer).

flow cell

access door

side door

data ports

optics

access door

sample
injection
tube

power button

fluidics cart

connections

The BD FACSCanto flow cytometer consists of a fluidics subsystem, an optics
subsystem, and an electronics subsystem. For a more in-depth discussion of

Chapter 1: Introduction 15

fluidics, optics, electronics, and flow cytometry, see the Technical Overview on
page 105.

Fluidics

The fluidics system consists of the sample injection tube (SIT), the aspirator arm,
the flow cell, a pressurized interior reservoir, and a network of tubing that
provides sheath and cleaning fluids to and removes waste from the flow cell.

flow cell

tubing

adapter lever

sample
injection
tube (SIT)

tube guide
(Loader option only)

aspirator arm

16 BD FACSCanto Flow Cytometer Reference Manual

aspirator arm bar

The following table briefly describes these components.

flow cell where laser beam intercepts particles

tubing brings sheath and cleaning fluids to and aspirates waste from flow cell

SIT hollow metal tube that brings sample to flow cell

adapter lever lever used to change SIT from manual to automatic loading

aspirator arm movable waste aspiration port

aspirator arm
bar

tube guide helps guide tube onto the SIT (Loader operation only)

metal bar used to push aspirator arm away from SIT during manual
loading

When you install tubes onto the SIT, a pump within the fluidics cart pressurizes
the interior reservoir (plenum), which then provides sheath fluid to the flow cell.
At the same time, the sample tube is pressurized and sample is pushed up the SIT
and into the flow cell.

When you remove tubes from the SIT, the cytometer cleans the SIT by flushing
sheath solution down the inside and outside of the tube. The flushed sheath
solution is aspirated by the aspirator arm.

To activate SIT cleaning, push the aspirator arm all the way to the left when you
manually unload a tube. When you are using the Loader, SIT cleaning occurs
automatically.

Do not leave a tube of distilled water on the SIT between sample tubes, or during
or after daily shutdown.

Chapter 1: Introduction 17

Flow Cell

Once the sample moves into the flow cell, particles move in single file past the
laser beams. The scattered and emitted light from these particles provides
information about their size, shape, granularity, and fluorescence properties.

The flow cell is beneath the flow cell access door. For more information, see
Fluidics System on page 106.

Figure 1-1 Flow cell

where lasers intercept

sample stream

obscuration bar

FSC diode

flow cell

Optics

The optics system in the BD FACSCanto cytometer is composed of both
excitation optics and collection optics. Excitation optics bring light to the flow
cell. Collection optics gather the light signals emitted or scattered by the particles.

Excitation Optics

The excitation optics consist of lasers, fiber optic cables, beam-shaping prisms,
and an achromatic focusing lens, as shown in Figure 1-2 on page 20.

18 BD FACSCanto Flow Cytometer Reference Manual

The BD FACSCanto cytometer uses low-powered air-cooled and solid state lasers
that do not have special power and cooling requirements.

Laser

Coherent® Sapphire™
Solid State

JDS Uniphase™ HeNe
Air Cooled

Wavelength

(nm)

488 (blue) FITC, PE, PerCP,

633 (red) APC, APC-Cy7

Commonly Used Fluorochromes

PerCP-Cy™5.5, PE-Cy™7

Chapter 1: Introduction 19

Fiber optic cables direct the laser light onto beam-shaping prisms, which in turn
transmit the laser light to a focusing lens. The lens directs the laser light onto the
sample stream within the flow cell (Figure 1-2). See also Figure A-3 on page 110.

Figure 1-2 Optical pathway

1

3

2

5

6

interlock flow cell

1

prisms red He-Ne laser

2 5

focusing lens blue solid-state laser

3 6

4

When the flow cell access door opens, an interlock shutters the laser light and
blocks its pathway for safety reasons.

4

20 BD FACSCanto Flow Cytometer Reference Manual

Collection Optics

From the flow cell, laser light is routed to the collection optics, which efficiently
gather the signals emitted and scattered from each particle. The BD FACSCanto
collection optics include two detector arrays, which consist of photomultiplier
tubes (PMTs) arranged in one octagon and one trigon (Figure 1-3).

The octagon contains five PMTs and detects light from the 488-nm (blue) laser.
One PMT in the octagon collects side scatter (SSC) signals.

The trigon contains two PMTs and detects light from the 633-nm (red) laser.

Figure 1-3 Octagon and trigon detector arrays

red-laser

A

585/42

488/10

E

signal

C

B

660/20

trigon

longpass

A

mirror

bandpass
filter

735LP

780/60

C

octagon

PMT

H

F

780/60

735LP

D

502LP

530/30

655LP

670LP

B

556LP

G

blue-laser

signal

Chapter 1: Introduction 21

When light arrives at an array, a longpass mirror (filter) transmits the highest
wavelengths to the first PMT in the series and reflects lower wavelengths to the
next PMT. Similarly, the next PMT’s longpass mirror transmits the next highest
wavelengths and reflect lower wavelengths, and so on around the array. A
bandpass filter (or additional longpass mirror) in front of each PMT further
screens unwanted light.

A

D

B

Fiber

E

In addition to the PMT detectors, a photodiode collects the stronger forward
scatter signals. The obscuration bar prevents excess laser light from entering this
diode (Figure 1-1 on page 18).

For additional descriptions of detector arrays, mirrors, and filters, see Optics
System on page 109.

22 BD FACSCanto Flow Cytometer Reference Manual

At installation, the octagon and trigon arrays have the filter and mirror
combinations shown in Table 1-1.

Table 1-1 Octagon and trigon optical filters

Detector Array

(Laser)

Octagon
(488-nm blue laser)

Trigon
(633-nm red laser)

PMT

Position

A 735 780/60 PE-Cy7

B 655 670 PerCP-Cy5.5 or

C 556 585/42 PE

D 502 530/30 FITC

Eblank

Fblank

Gblank

H— blank

A 735 780/60 APC-Cy7

B blank 660/20 APC

LP Mirror

optical holder

optical holder

optical holder

BP Filter or

LP Mirror

488/10 SSC

blank

optical holder

blank

optical holder

optical holder

Intended Dye

PerCP

—

—

—

Blank optical holders do not contain optical filters. They are used in the octagon
and trigon to prevent unwanted light from interfering with fluorescence signal.

Chapter 1: Introduction 23

Electronics

The electronics system converts optical signals to electronic signals and digitizes
them for computer analysis. The photodiode and PMTs generate signals
proportional to the amount of light they detect. The cytometer’s onboard
electronics amplifies and then converts the signals from continuous voltage
values (analog) into discrete values (digital). Upon amplification and digital
conversion, fluorescent light signals from consistently prepared and stained
particles characteristically fall into certain channels, thus allowing analysis.

On the BD FACSCanto, electronic system components consist of power controls
and connectors along with processing boards in the card cage. This section
describes only the user-accessible power panel. For more information, see
Electronics System on page 120.

Power Panel

Power to the cytometer, lasers, and fluidics cart is supplied by a power cord from
the cytometer plugged directly into a standard electrical outlet. BD recommends
using an uninterrupted power supply (UPS) unit to maintain cytometer power
during a power outage. The system power button turns on the cytometer and
fluidics cart, and powers the lasers (Figure 1-4).

Figure 1-4 Flow cytometer power panel

system AC power

cord plugs in here

system circuit breaker

The system circuit breaker is located next to the AC power cord. The breaker will
need to be reset if there is a power surge in the laboratory.

24 BD FACSCanto Flow Cytometer Reference Manual

system power
button

Fluidics Cart

The fluidics cart provides filtered sheath and cleaning fluids to the cytometer, and
collects system waste products (Figure 1-5). The cart supplies the required air
pressure and vacuum, which eliminates the need for an external source (although
the cart can be hooked up to an in-house air source).

Figure 1-5 Fluidics cart

cubitainer

10-L waste
container

pressure

gauge

door access

knob

filters

Chapter 1: Introduction 25

Containers and Ports

The fluidics cart holds a 10-L waste container, a 20-L BD FACSFlow™
cubitainer, a 5-L BD FACS shutdown solution cubitainer, and a 5-L
BD FACSClean solution cubitainer.

Use the waste container provided with the system; do not substitute other
containers.

Figure 1-6 Fluidics cart containers and ports

BD FACS™ shutdown
solution port and cubitainer

waste port

BD™ FACSClean port and
cubitainer

BD FACSFlow™ port

waste tank

BD FACSFlow cubitainer

Each solution has its own non-interchangeable fluid port and level-sensor
connection. Fluid level alarms occur within BD FACSCanto clinical software and
BD FACSDiva software.

26 BD FACSCanto Flow Cytometer Reference Manual

Controls

The fluidics cart connects to the flow cytometer unit by way of cables and tubing.
When you turn on the power to the cytometer, the fluidics cart powers on also.
Under ordinary circumstances, you do not need to adjust any of the switches on
the cart’s power panel. Leave the auxiliary air supply switch off unless the cart
has been attached to an in-house air supply by BD Biosciences service personnel
(see Table 1-2 for details). Leave the cart circuit breaker on at all times.

Figure 1-7 Fluidics cart panel

auxiliary air supply
switch, off

cart circuit breaker, on

Table 1-2 Auxiliary air supply switch positions

Position Air Source What This Means

Off Cart air The cart is providing its own air

pressure and vacuum.

On House air The cart is hooked up to the

building’s air pressure and vacuum.

Chapter 1: Introduction 27

Powering Off

To turn off the fluidics cart (and the cytometer, as well), press the system power
button. Normally during cart shutdown, you hear a hiss, and a small amount of
condensed water will discharge.

Computer Workstation

The workstation consists of a computer compatible with Microsoft Windows XP
Professional operating system, a flat-screen monitor, and an optional printer.

BD FACS Loader (Optional)

The BD FACS Loader automatically introduces prepared samples to the
cytometer. The Loader is controlled by BD FACSCanto clinical software or
BD FACSDiva software. It can be added to your system at any time. The Loader
consists of:

•a drawer

• a cover

• three optical sensors

• an electronics module

•a tube lifter

• a 40-tube carousel

For more information, see BD FACS Loader Option on page 33.

28 BD FACSCanto Flow Cytometer Reference Manual

Figure 1-8 BD FACS Loader

Chapter 1: Introduction 29

Barcode Reader (Optional)

The Opticon LG2 Imager is a hand-held barcode reader that plugs into the USB
port on the BD FACSCanto computer workstation. A green light on the top of
the barcode reader indicates that it is connected to the USB port and ready to use.

The barcode reader reads most barcode standards, including Codabar, Code 128,
Code 39 with checksum, and PDF417. It reads information from the BD FACS™
7-color setup beads label into BD FACSCanto clinical software, and also reads
coded patient information into a worklist.

Figure 1-9 Typical barcode reader (example)

ready light

trigger

view from top

For information on installing and using the barcode reader with the
BD FACSCanto, see Installing and Using the Barcode Reader on page 46. For
detailed information including how to program the barcode reader for other
barcode standards, refer to the information supplied by the manufacturer.

30 BD FACSCanto Flow Cytometer Reference Manual

System Requirements

Software

Both included software packages must be installed:

• BD FACSCanto clinical software v2.1

Do not read FCS files created with v2.0 or v2.1 into previous versions of
BD FACSCanto clinical software. Previous versions will show incorrect results.

• BD FACSDiva software v6.0

NOTICE BD FACSCanto clinical software v2.0 can be used to operate the
BD FACSCanto system with BD FACSDiva™ software v5.0 installed.
However, to use BD FACSDiva™ software v6.0, BD FACSCanto clinical
software v2.1 must be installed.

Workstation

BD FACSCanto workstation purchased through BD Biosciences

Compatible Tubes

• 12 x 75-mm polystyrene test tubes (BD Falcon™ tubes)

• 12 x 75-mm BD Trucount™ tubes

• BD FACS™ 7-color setup bead tubes

Bulk Fluids

• BD FACSFlow solution

• BD™ FACSClean solution

• BD FACS™ shutdown solution

Chapter 1: Introduction 31

• full-strength bleach (waste tank)

Fluids Required for External Cleaning

• BD FACSClean solution

• Deionized (DI) water

Setup Beads

BD FACS 7-color setup beads for use with BD FACSCanto clinical software

32 BD FACSCanto Flow Cytometer Reference Manual

BD FACS Loader Option

This chapter contains the following information:

• Loader Components on page 34

• Using the Loader on page 36

• Maintaining the Loader on page 43

2

33

Loader Components

The Loader is mounted directly on the flow cytometer. The device consists of a
drive system, tube lifter mechanism, spindle, and optical sensors, all of which are
attached to a sliding drawer. A cover fits over the drawer to protect you from
moving parts during operation. Table 2-1 provides a brief description of Loader
components.

tube guide

alignment guide pin

tube optical sensor

carousel optical sensor

tube lifter

drawer

center spindle

Table 2-1 Loader components

tube guide guides tubes during lifting to ensure a proper seal

alignment guide pin aligns the carousel and keeps it in place

carousel optical sensor reads the carousel ID

tube optical sensor scans the carousel to verify tube locations and

match the associated worklist, and verifies a tube
is in place before activating the tube lifter

tube lifter a stainless steel rod that lifts the sample tube

from the carousel to the sample injection tube
(SIT)

34 BD FACSCanto Flow Cytometer Reference Manual

Table 2-1 Loader components (continued)

drawer holds the carousel; slides out for easy access

center spindle the axis around which the carousel spins; insert

the carousel onto the Loader here

drive system the motor that rotates the carousel (not visible

from exterior)

Carousel

The carousel accommodates up to forty 12 x 75-mm tubes. Each carousel has a
unique ID printed on top and on an optically read label inside.

Figure 2-1 Sample-Prep Ready carousel

spindle hole

alignment hole

The Loader is compatible with both the green-tinted carousels labeled “Sample­Prep Ready” and the traditional gray Loader carousels (Figure 2-2).

Figure 2-2 Compatible carousels

Sample-Prep Ready carousel traditional gray carousel

Chapter 2: BD FACS Loader Option 35

Not all manufactured 12 x 75-mm tubes have been checked for proper
functionality on the Loader. BD Biosciences has validated only disposable,
12 x 75-mm BD Falcon polystyrene test tubes, BD Trucount tubes, and
BD FACS 7-color setup bead tubes.

Using the Loader

The Loader operates by software commands only. Commands can be issued with
either BD FACSCanto clinical software or BD FACSDiva software. For
instructions on running samples with the Loader, refer to the BD FACSCanto
Instructions for Use.

This section explains how to set up the cytometer for acquisition with the Loader.

1 Verify that the cytometer is set up for automatic loading.

The adapter lever should be in the lowest position, the tube guide should be
forward, and the aspirator arm bar should be horizontal (Figure 2-3). For
detailed instructions, see Changing to Automatic Loading on page 41.

Figure 2-3 Automatic loading setup

adapter lever

tube guide

aspirator arm bar

36 BD FACSCanto Flow Cytometer Reference Manual

2 If needed, remove the carousel from the Loader:

• Open the Loader cover.

• Pull out the Loader drawer.

• Place your thumb on the center spindle, and press down with your

thumb while gently pulling up on the carousel handle with two fingers,
as shown.

3 Vortex the sample tubes and place them uncapped in the carousel(s)

according to the worklist.

For accurate results, match the tubes to those listed on the printed worklist
(BD FACSCanto clinical software) or the Carousel Assignment tab
(BD FACSDiva software). Print a copy of tube assignments and use the
printout as a guide when filling each carousel.

To prevent binding during loading, make sure label thickness per tube is
less than three labels. Flatten labels completely before placing a tube in the
carousel.

Chapter 2: BD FACS Loader Option 37

4 Install the first carousel to be acquired on the Loader.

Position the spindle hole in the handle over the center spindle of the Loader
drawer. Rotate the carousel until the alignment guide pin fits into the small
alignment hole at the top of the carousel. Press down firmly to seat the
carousel.

Figure 2-4 Seating the carousel in the Loader

spindle

handle

alignment pin

5 Close the Loader drawer completely, and install the Loader cover.

The Loader scans and positions the carousel at tube 1.

To run the Loader, the cover must be in place on the drawer. Tubes will not
be loaded if the cover is off. The currently running tube will be unloaded if
the cover is removed during a run.

Loader cover

38 BD FACSCanto Flow Cytometer Reference Manual

Changing Operational Modes

Performing a few simple steps allows you to change from automatic loading to
manual loading. See the following sections for instructions.

• Changing to Manual Loading (on this page)

• Changing to Automatic Loading on page 41

Changing to Manual Loading

To change to manual mode, follow these steps.

Handle all biological specimens and materials as if capable of transmitting
infection. Dispose of waste using proper precautions and in accordance
with local regulations. Wear suitable protective clothing, eyewear, and
gloves.

Materials

personal protective equipment

Procedure

1 Remove the Loader cover.

2 Pull out the drawer.

3 Remove any carousel in the Loader.

Chapter 2: BD FACS Loader Option 39

4 Move the tube guide arm all the way back and away from you (in manual

mode, you do not need the tube guide arm).

automatic mode

manual mode

5 Lift the adapter lever and rotate it into the highest position, as shown in the

following figure.

adapter lever,
manual mode

adapter lever,
automatic mode

6 (Optional) Rotate the aspirator arm bar vertically.

automatic mode manual mode

You are ready to load tubes manually.

40 BD FACSCanto Flow Cytometer Reference Manual

Changing to Automatic Loading

To change from manual loading to automatic loading, follow these steps.

Handle all biological specimens and materials as if capable of transmitting
infection. Dispose of waste using proper precautions and in accordance
with local regulations. Wear suitable protective clothing, eyewear, and
gloves.

Materials

personal protective equipment

Procedure

1 Lift the adapter lever and rotate it into the automatic mode position, as

shown in the following figure.

adapter lever,
manual mode

adapter lever,
automatic mode

Chapter 2: BD FACS Loader Option 41

2 Rotate the aspirator arm bar to the horizontal position until it reaches the

stop pin.

automatic modemanual mode

3 Move the tube guide arm all the way forward.

The tube guide arm is needed only when using the Loader.

manual mode

automatic mode

4 Pull out the drawer.

You are ready to load tubes automatically.

42 BD FACSCanto Flow Cytometer Reference Manual

Maintaining the Loader

This section explains the cleaning procedure you should follow to keep your
Loader in good condition.

Cleaning External Surfaces

To keep the Loader free from salt buildup, wipe down all external surfaces that
have been exposed to sheath fluid.

All cytometer surfaces that come in contact with biological specimens can
transmit potentially fatal disease. Use universal precautions when cleaning
cytometer surfaces. Wear suitable protective clothing and gloves.

To prevent damage, do not use isopropyl alcohol or ethanol on any Loader
surfaces.

Materials

• BD FACSClean solution

•DI water

• clean, lint-free cloths or disposable wipes

Chapter 2: BD FACS Loader Option 43

Procedure

To avoid potential shock, always switch off the power and unplug the AC
power cord before you begin cleaning.

1 Switch off the cytometer power and unplug the AC power cord from the

wall socket.

2 Wipe all accessible surfaces with BD FACSClean solution.

Ensure that the optical sensors are cleaned. Saline deposits can develop on
the sensors, causing tubes in the carousel to be missed.

3 Wet a fresh cloth with DI water and wipe all exposed surfaces to prevent

corrosion.

4 Wipe all exposed surfaces with a clean, dry cloth.

44 BD FACSCanto Flow Cytometer Reference Manual

Barcode Reader Option

This chapter contains the following information:

• Installing and Using the Barcode Reader on page 46

• Cleaning the Barcode Reader on page 48

• Barcode Symbologies on page 49

3

45

Installing and Using the Barcode Reader

To prevent laser injury, do not stare into the reader or aim it at another
person’s eyes while the trigger is depressed.

NOTICE The Opticon LG2 Imager2D barcode reader available for the
BD FACSCanto is capable of reading both 1D and 2D barcode symbologies. See
Barcode Symbologies on page 49.

The BD FACSCanto system supports only two uses of the 2D barcode reader:

• reading information from the BD FACS 7-color setup beads label into
BD FACSCanto clinical software (2D barcode symbology)

• reading coded patient information from sample tube labels into a worklist
(1D barcode symbology)

NOTICE The barcode reader supplied with the BD FACS™ Sample Prep
Assistant II (SPA II) is a 1D reader that cannot read information from
BD FACS 7-color setup beads. Use only the Opticon LG2 Imager 2D barcode
reader for reading information from the BD FACS 7-color setup beads label into
BD FACSCanto clinical software.

NOTICE Contact BD Biosciences before changing default barcode settings.

46 BD FACSCanto Flow Cytometer Reference Manual

1 Plug the barcode reader into the USB port on the back of your computer.

The reader turns on automatically.

2 Make sure the green light on top of the reader is illuminated:

green light

3 In BD FACSCanto clinical software, navigate to the fields for which

information can be scanned.

• Lot information for BD FACS 7-color setup beads is scanned into the
Setup Lot Information dialog from the setup values label supplied with
the setup bead kit (NOT from the lot number on the setup bead tubes).

• Patient information is scanned from sample labels into the worklist ID
field.

4 Locate the barcode on the label of BD FACS 7-color setup beads kit, or on

the patient sample.

For accurate results, do not photocopy or enlarge the barcodes that are
included with the reagent. Scan the barcodes exactly as they are provided.

; Tip To make it easier to use, an optional stand is available for the barcode

reader. See Bar Code Reader Parts on page 129.

Chapter 3: Barcode Reader Option 47

5 Aim the barcode reader at the center of the barcode. The barcode reader

has two focal lengths, 4 and 9 inches:

• For reading BD FACS 7-color setup bead labels, hold the barcode
reader 9 inches from the labels.

• For reading patient information from sample tube labels, hold the
barcode reader 4 inches from the labels.

6 Press and hold the trigger on the barcode reader until you hear a beep.

If the reader does not beep, adjust your distance from the barcode while
continuing to hold the trigger.

; Tip To obtain a reading, keep the bar code reader aimed at the center of

the label. Do NOT sweep across the label.

7 Compare software field values with the setup beads or sample label.

Cleaning the Barcode Reader

To prevent laser injury, do not stare into the reader or aim it at another
person’s eyes while the trigger is depressed.

For best performance, keep the front window of the barcode reader clean. Do not
touch the window directly, and wipe it only with a soft, non-abrasive cloth
moistened with one of the following:

• isopropyl alcohol

• ethyl alcohol (denatured grade)

Do not use BD FACSClean solution (or bleach) to clean or disinfect the
barcode reader.

48 BD FACSCanto Flow Cytometer Reference Manual

Barcode Symbologies

Although data entry using barcodes is generally more reliable than manual data
entry, it is not guaranteed to be 100% accurate. To increase accuracy when using
the barcode reader, enabling checksums is recommended.

Using barcode symbologies without checksums increases the likelihood of
incorrect information transfer, including sample ID assignments. This can
result in a mismatch of sample IDs and sample results.

By default, the barcode reader has checksums enabled. We recommend you
do not disable checksums, or use barcode symbologies without checksums.

1D Barcode Symbologies

BD Biosciences has evaluated the following 1D barcode symbologies for use with
the BD FACSCanto flow cytometer, and has these recommendations:

Barcode Symbology Recommendation

Code 128 Preferred.

Code 39 Acceptable if barcode labels are printed with the checksum

digit. By default, the barcode reader recognizes the checksum
digit when reading the Code 39 symbology. However, if labels
are printed without a checksum digit, contact your BD service
representative for instructions on disabling the checksum
feature.

Codabar The barcode reader does not support the checksum feature

when reading the Codabar symbology.

Chapter 3: Barcode Reader Option 49

2D Barcode Symbologies

BD Biosciences has evaluated 2D barcode symbology to read the target values of
BD FACS 7-color setup beads when using BD FACSCanto clinical software. 2D
barcode symbology is required to read all target values with one scan.

For information on installing and using the barcode readInstalling and Using the
Barcode Reader on page 46er with the BD FACSCanto system, see Installing and
Using the Barcode Reader on page 46. For detailed information including how to
program the barcode reader for other barcode standards, refer to the information
supplied by the manufacturer.

50 BD FACSCanto Flow Cytometer Reference Manual

4

Maintenance

The BD FACSCanto system requires basic preventive maintenance to preserve
cytometer reliability. This section explains the procedures you should follow to
keep your cytometer in good condition.

• Scheduled Maintenance on page 52

• Unscheduled Maintenance on page 66

51

Scheduled Maintenance

For optimal cytometer functioning, perform the following maintenance
according to the recommended schedule.

Procedure Description and Location Frequency

Fluidics startup Replaces BD FACS shutdown

solution with BD FACSFlow
solution

Refer to the BD FACSCanto

Instructions for Use.

Fluidics shutdown Replaces BD FACSFlow

solution with
BD FACS shutdown solution

Refer to the BD FACSCanto

Instructions for Use.

Wipe down SIT and
aspirator arm

Empty the waste See Emptying the Waste

Purge fluid filters Removes air from fluid filters,

Change the waste tank
cap

Prevents saline deposit buildup

Refer to the BD FACSCanto

Instructions for Use.

Container on page 54.

ensuring they will not dry out

See Purging the Fluidics Filters
on page 57.

See step 6 on page 56 (in
Emptying the Waste Container
procedure).

Daily

Daily

Daily

Daily and as needed

Weekly

Monthly

52 BD FACSCanto Flow Cytometer Reference Manual

Procedure Description and Location Frequency

Decontaminate fluidics
(Long Clean)

Replace the air filter Ensures proper cytometer

Replace fluid filters Keeps fluids free of particulates

Schedule preventative
maintenance by BD
service

Decontaminates the internal
sheath path with
BD FACSClean solution, then
rinses with BD FACS shutdown
solution

See Decontaminating the
Fluidics System (Long Clean) on
page 59.

performance

See Replacing the Air Filter on
page 60.

See Replacing Fluidics Filters on
page 63.

— Every 6 months

Monthly and before
service calls

Every 6 months

Every 6 months or when
increased debris observed
in FSC vs SSC plots

Chapter 4: Maintenance 53

Emptying the Waste Container

All biological specimens and materials coming into contact with them can
transmit potentially fatal disease. To prevent exposure to biohazardous
agents, expose waste container contents to bleach (10% of total volume)
before disposal. Dispose of waste in accordance with local regulations. Use
proper precaution and wear suitable protective clothing, eyewear, and
gloves.

waste port

disposable

waste cap

trap

waste tank

1 Ensure the cytometer is not acquiring events.

54 BD FACSCanto Flow Cytometer Reference Manual

2 Detach the waste container’s sensor and fluid line.

• Pull the sensor straight out.

• Press the metal clip on the fluid line.

waste fluid line

sensor

The waste tank can become pressurized when the cytometer is running.
Always disconnect the tank from the fluidics cart before you empty it. Wait
at least 30 seconds for pressure to dissipate before you remove the waste
cap or level sensor cap.

with metal clip

3 Remove the disposable waste cap and attached trap from the container.

Place the assembly on the bench label-side up.

Do not wet the cap. If you see liquid inside the trap, remove the drain plug
and fully drain the liquid before you replace the plug.

trap

drain plug

Chapter 4: Maintenance 55

4 Empty the bleach-exposed waste.

5 Add approximately 1 L of bleach to the empty waste container (10 L

container).

6 If one month has passed since the last cap change:

• Detach the cap from the trap.

• Replace it with a new cap.

• Write the date on the cap label.

Waste (A)

338677 Rev A

To prevent tank overpressurization, do not overtighten the trap or attached
filter cap. Tighten each component only until it is hand-tight. Do not use
sealants such as Teflon® tape or other adhesives.

space for date

7 Screw the cap assembly onto the tank and hand-tighten until it is fully

closed.

8 Re-attach the sensor and fluid lines.

56 BD FACSCanto Flow Cytometer Reference Manual

Purging the Fluidics Filters

Once a week, purge air from all fluid filters by opening the bleeder valve on the
top of each filter, one at a time. This ensures that the filters will not dry out.

NOTICE The cytometer must be on (system pressurized) during this procedure.

Materials

• paper towels

• proper protective equipment

Procedure

1 Run the Prime After Tank Refill for all fluids to ensure fluid lines are

primed.

See Priming Fluidics Lines on page 71.

2 Place a few paper towels beneath the filter to be purged to absorb any fluid

leakage.

Chapter 4: Maintenance 57

3 Loosen the bleeder valve near the top of the filter by turning it

counterclockwise.

NOTICE Do not loosen the bottom bleeder valve. Ensure it is tightened.

; Tip Do not completely unscrew the valve. If you do, it will come off.

loosen this
bleeder valve

DO NOT loosen
this bleeder valve

4 Wait until fluid seeps out.

Fluid should seep from the open valve within 30 seconds. If no fluid
appears, make sure the corresponding cubitainer is not empty or detached
from the cart. If the cubitainer contains fluid and the fluid lines are
attached and primed, the filter might be airlocked. See Removing an Air
Lock on page 72.

5 Close the valve by turning it clockwise.

6 Repeat steps 2 through 5 with the remaining filters.

58 BD FACSCanto Flow Cytometer Reference Manual

Decontaminating the Fluidics System (Long Clean)

Use the Long Clean command to decontaminate the internal sheath path with
BD FACSClean solution solution. After decontamination, the lines are rinsed
with BD FACS shutdown solution.

NOTICE This procedure takes 75 minutes to complete.

Materials

• undiluted bleach (for waste tank)

• BD FACSClean solution (approximately 275 mL)

• BD FACS shutdown solution (approximately 1,100 mL)

Procedure

1 Check all fluid levels; empty the waste if needed.

2 Select Cytometer > Cleaning Modes > Long Clean.

A confirmation dialog displays.

3 Click OK to continue. Once you have begun the Long Clean, you cannot

abort the process.

A completion message displays when the cleaning cycle finishes.

Figure 4-1 Long Clean completion message

BD FACSCanto clinical software

BD FACSDiva software

Chapter 4: Maintenance 59

4 Click OK.

If the completion message does not display after 90 minutes, verify there
are no error messages in the Status tab of the Cytometer window. If the
cleaning mode fails, see Fluidics Cart Troubleshooting on page 101.

5 Select to shut down or continue.

• To shut down, select Cytometer > Fluidics Shutdown.
Repeat Fluidics Shutdown twice more.
Quit the software and turn off the power to the cytometer.

• To continue, select Cytometer > Fluidics Startup.
Repeat Fluidics Startup twice more.

BD FACS shutdown solution can lyse cells. The Fluidics Startup
procedure removes BD FACS shutdown solution from the interior
reservoir and fluid lines and replaces it with BD FACSFlow solution.

; Tip If setup fails after a long clean, repeat Fluidics Startup until setup

passes.

Replacing the Air Filter

The BD FACSCanto cytometer has an air filter located in the side door. Replace
the filter every 6 months to ensure proper cytometer performance.

Materials

Replacement filter (see Other Replacement Parts on page 129 for ordering)

Procedure

1 Turn off the cytometer power.

2 Open the side door.

• Press the black button; a handle will pop out.

60 BD FACSCanto Flow Cytometer Reference Manual

• Twist the handle and pull.

3 On the door’s interior, turn the pegs along the upper edge of the filter and

remove the old filter.

Chapter 4: Maintenance 61

Dispose of the old filter. It cannot be reused.

4 Install a new filter, making sure the arrow on top of the filter points in

toward the cytometer (arrow indicates direction of air flow).

5 Turn the pegs to hold the filter in place.

6 Close the side door:

• Twist the handle while closing door.

• Push door closed until the door latches.

• Push the handle in to latch the handle.

To avoid cytometer damage, do not close the door on any tubing or wires.

62 BD FACSCanto Flow Cytometer Reference Manual

Replacing Fluidics Filters

Change the fluid filters when you see increased debris in FSC vs SSC plots, or
every 6 months.

filter

Materials

• replacement filter(s)—see Installation Kit on page 128 for ordering

• paper towels

• proper protective equipment

• felt tip pen

Chapter 4: Maintenance 63

Procedure

1 Place a few paper towels beneath the filter to collect drips.

2 Remove the filter by pressing the tabs on each quick-disconnect coupling.

Figure 4-2 Removing the filter

metal tab

quick-disconnect

coupling

metal tab

3 Position the new filter and connect the couplings.

; Tip Write today’s date on the filter so you will know when to replace it.

64 BD FACSCanto Flow Cytometer Reference Manual

4 Loosen the top bleeder valve by turning it counterclockwise.

NOTICE Do not loosen the bottom bleeder valve. Ensure it is tightened.

; Tip Do not completely unscrew the valve. If you do, it will come off.

loosen this
bleeder valve

DO NOT loosen
this bleeder valve

5 Wait until fluid seeps out.

Fluid should seep from the open valve within 30 seconds. If no fluid
appears, ensure the corresponding cubitainer is not empty or detached from
the cart. If the cubitainer contains fluid and the fluid lines are attached and
primed, the filter might be airlocked. See Removing an Air Lock on
page 72.

6 Close the valve by turning it clockwise.

Repeat the procedure for other fluid filters to be replaced.

Chapter 4: Maintenance 65

Unscheduled Maintenance

Perform these maintenance procedures as needed.

Procedure Description and Location Frequency

Replace cubitainers See Changing a Cubitainer on

page 68.

Prime fluid lines Removes air from fluid lines

See Priming Fluidics Lines on
page 71.

Remove an air lock Removes air from filter and

restores flow of fluid

See Removing an Air Lock on
page 72.

Clean external surfaces Keeps surfaces free from saline

deposit buildup after exposure
to sheath fluid

See Cleaning External Surfaces
on page 75.

De-gas Flow Cell Removes bubbles from the flow

cell

See Removing Bubbles from the
Flow Cell on page 76.

Clean Flow Cell Runs BD FACSClean solution

through the SIT and flow cell

See Cleaning the Flow Cell on
page 77.

As needed

Whenever a fluidics line
is disconnected to change
a cubitainer or perform
other maintenance

When fluidics are not
functioning properly
(flow cell will not fill, or
there is backflush into
sample tube)

As needed

As needed during daily
startup

When poor optical
performance indicates
additional cleaning is
needed

Bubble Filter Purge Removes air from the bubble

filter

See Purging the Bubble Filter on
page 78.

66 BD FACSCanto Flow Cytometer Reference Manual

If fluidics run dry, or
when CVs are poor

Procedure Description and Location Frequency

Decontaminate the
fluidics system for
storage

Replace the Bal seal Replaces worn Bal seal

Reset the cytometer
circuit breaker

Reattach Ethernet and
RS232 cables

Reconnect fluidics cart
tubing

Replace fluidics level
sensors

Replace fluidics cart fuses See Replacing the Fluidics Cart

Cleans out the fluidics lines with
BD FACSClean solution, then
fills them with
BD FACS shutdown solution

See Decontaminating the
Fluidics System for Storage on
page 80.

See Replacing the Bal Seal on
page 80.

See Resetting the Cytometer
Circuit Breaker on page 84.

See Reconnecting the Ethernet
and RS232 Cables on page 85.

See Reconnecting the Fluidics
Cart Tubing on page 85.

See Replacing the Fluidics Level
Sensors on page 88.

Fuses on page 91.

Before long-term storage

When the seal becomes
worn or cracked (sample
tubes will not pressurize)

After the circuit breaker
is tripped

When a cable is
disconnected

As needed

When instructed to by a
BD Biosciences service
representative

When cart will not
function because of power
surge or other electrical
event

Chapter 4: Maintenance 67

Changing a Cubitainer

Three fluidics cubitainers (disposable boxes of approved fluids) and a waste
container fit onto the cart in the following configuration:

BD FACS shutdown solution

waste container

BD FACSClean solution

BD FACSFlow solution

Each cubitainer and the waste tank has its own color-coded port. Connect
containers to the following ports:

Container Port Label Port Color

waste Waste (A) orange-red

BD FACSFlow solution BD FACSFlow blue

BD FACS shutdown solution BD FACS Shutdown Solution green

BD FACSClean solution BD FACS Cleaning Solution green

68 BD FACSCanto Flow Cytometer Reference Manual

Procedure

1 Ensure the cytometer is not acquiring events.

2 Detach the sensor and fluid line from the cart.

• Pull the sensor straight out.

sensor

• Press the metal clip on the fluid line.

metal clip

You could damage the sensor line if you leave it connected while changing a
cubitainer.

3 Unscrew the cap on the cubitainer.

Chapter 4: Maintenance 69

4 Remove the cap and sensor assembly and set it aside.

I

5 Put a new cubitainer onto the fluidics cart.

6 Replace the cap assembly and hand-tighten it until it is fully closed.

7 Reattach the sensor line and fluid line to the cart.

• To attach the sensor line, gently rotate until the connection aligns, and
then push.

• To attach the fluid line, push the coupling into the port until it clicks.

To ensure that the appropriate solutions are dispensed, match the label on
the container to the port on the fluidics cart.

8 Prime the fluidics.

Important: Continue with the procedure in Priming Fluidics Lines on
page 71.

70 BD FACSCanto Flow Cytometer Reference Manual

Priming Fluidics Lines

Use the Prime After Tank Refill command to remove air from the fluid lines after
you change a cubitainer or detach the fluidics lines for other maintenance.

1 Select Cytometer > Cleaning Modes > Prime After Tank Refill.

2 Select the checkboxes for the cubitainers you changed; click OK.

BD FACSDiva softwareBD FACSCanto clinical software

3 Click OK when the completion message displays.

BD FACSCanto clinical software

BD FACSDiva software

Chapter 4: Maintenance 71

Removing an Air Lock

If too much air gets into the sheath filter, it becomes impermeable to fluid and an
air lock can develop. Although it is most likely to occur for the sheath filter, an
air lock can develop in any of the fluidics filters.

All cytometer surfaces that come in contact with biological specimens can
transmit potentially fatal disease. Use universal precautions when cleaning
cytometer surfaces. Wear suitable protective clothing and gloves.

Materials

• paper towels

• bypass tubing

Procedure

1 Place a few paper towels beneath the air-locked filter to collect drips.

2 Remove the filter by pressing the metal tabs.

Figure 4-3 Removing a filter

metal tab

metal tab

72 BD FACSCanto Flow Cytometer Reference Manual

3 Install bypass tubing in place of the filter.

Push the tubing into each quick-disconnect port until you hear a click.

bypass tubing,
installed

4 Select Cytometer > Cleaning Modes > Prime After Tank Refill.

5 Select the checkbox that corresponds to the filter you have bypassed.

6 Click OK.

BD FACSCanto clinical software

BD FACSDiva software

7 When the prime finishes, remove the bypass tubing.

8 Reattach the filter to the fluidics cart.

9 Repeat the Prime After Tank Refill command.

Chapter 4: Maintenance 73

10 Open the bleeder valve and wait for fluid to seep out. Close the valve.

If no fluid seeps out, repeat steps 9 and 10.

Figure 4-4 Bleeder valve

loosen this
bleeder valve

DO NOT loosen
this bleeder valve

74 BD FACSCanto Flow Cytometer Reference Manual

Cleaning External Surfaces

To prevent salt buildup, wipe down all external cytometer surfaces that have
been exposed to sheath fluid.

NOTICE Over time, saline deposits may develop on the interior surface of the
SIT Bal seal retainer, causing an incomplete SIT flush between samples. If this
occurs, you might need to remove and clean the retainer with DI water. See
Figure 4-5 on page 80 and steps 4, 7, and 8 of the Replacing the Bal Seal
procedure that starts on page 80.

NOTICE Do not use BD FACSClean solution or bleach to clean or disinfect the
barcode reader. See Cleaning the Barcode Reader on page 48.

All cytometer surfaces that come in contact with biological specimens can
transmit potentially fatal disease. Use universal precautions when cleaning
cytometer surfaces. Wear suitable protective clothing and gloves.

To prevent damage, do not use isopropyl alcohol or ethanol on any
cytometer or fluidics cart surface.

Materials

• BD FACSClean solution

•DI water

• clean, lint-free cloths or disposable wipe

Chapter 4: Maintenance 75

Procedure

1 Switch off the cytometer power and unplug the AC power cord.

To avoid potential shock, always switch off the power and unplug the AC
power cord before you begin cleaning.

2 Wipe all accessible surfaces with BD FACSClean solution.

3 Wet a fresh cloth with DI water and wipe all bleach-exposed surfaces to

prevent corrosion.

4 Wipe all exposed surfaces with a clean, dry cloth.

Removing Bubbles from the Flow Cell

Use the De-gas Flow Cell command to remove bubbles from the flow cell.

1 Select Cytometer > Cleaning Modes > De-gas Flow Cell.

2 Click OK when the completion message displays.

BD FACSCanto clinical software

BD FACSDiva software

3 Check the flow cell for bubbles.

If you still see bubbles, repeat the process.

76 BD FACSCanto Flow Cytometer Reference Manual

Cleaning the Flow Cell

Use the Clean Flow Cell command to run a tube of BD FACSClean solution
through the SIT and flow cell. After the procedure is complete, the cleaning fluid
remains in the SIT and flow cell until you run fluidics startup or shutdown.
Perform this procedure when poor optical performance indicates that additional
cleaning is needed.

Materials

• 3 mL of BD FACSClean solution

• one 12 x 75-mm polystyrene BD Falcon tube

Procedure

1 Select Cytometer > Cleaning Modes > Clean Flow Cell.

2 If you have a Loader, remove the carousel.

3 When prompted, manually install a tube containing approximately 3 mL of

BD FACSClean solution onto the SIT, and click OK.

BD FACSCanto clinical software

BD FACSDiva software

Chapter 4: Maintenance 77

A progress message displays during the cleaning.

BD FACSCanto clinical software

BD FACSDiva software

4 After the completion message displays, wait 5 minutes.

Waiting allows the BD FACSClean solution to dissolve deposits in the flow
cell cuvette.

5 Click OK.

6 Remove the tube from the SIT.

7 Clear BD FACSClean solution from the flow cell and fluidics lines:

• Before running setup or samples, run Fluidics Startup.

• If you are shutting down without running more samples, run Fluidics
Shutdown.

Purging the Bubble Filter

The bubble filter sits between the fluidics tanks and reservoirs and the flow cell
and ensures that the flow cell remains bubble-free. However, should the fluidics
run dry, you might need to remove air from the bubble filter. Poor CVs might
indicate a need to purge the bubble filter.

1 Select Cytometer > Cleaning Modes > Bubble Filter Purge.

78 BD FACSCanto Flow Cytometer Reference Manual

2 A dialog indicates Bubble Filter Purge progress. Wait until the purge

finishes.

BD FACSCanto clinical software

BD FACSDiva software

3 Click OK when the completion message is displayed.

BD FACSCanto clinical software

BD FACSDiva software

4 Repeat steps 1 through 3 until bubble-free liquid enters the flow cell from

the bubble filter.

5 When finished, select Cytometer > Cleaning Modes > De-gas Flow Cell.

Chapter 4: Maintenance 79

Decontaminating the Fluidics System for Storage

If your cytometer is to be taken out of operation, perform the following
procedure to clean out the fluidics lines and fill them with BD FACS shutdown
solution.

1 Perform steps 1 through 4 in Decontaminating the Fluidics System (Long

Clean) on page 59.

2 Run fluidics shutdown.

3 Shut down the software and turn off the power to the cytometer.

Replacing the Bal Seal

The Bal seal is a Teflon ring that allows the sample tube to pressurize. Over time,
this seal becomes worn or cracked and requires replacement. Replace the seal
when persistent sample tube pressure errors occur.

Over time, saline deposits may develop on the interior surface of the SIT Bal seal
retainer, causing an incomplete SIT flush between samples. Therefore, you should
always rinse the retainer in DI water when you replace a Bal seal.

Figure 4-5 Bal seal and retainer

Bal seal

SIT

80 BD FACSCanto Flow Cytometer Reference Manual

retainer

Materials

• proper protective equipment

• replacement Bal seal

•DI water

Procedure

1 Turn off the cytometer.

2 If you have a Loader, follow these steps:

• Remove the Loader cover.

• Pull out the drawer.

• Remove any carousel.

• Change the cytometer from automatic to manual mode (see Changing

to Manual Loading on page 39).

If you do not change the cytometer’s mode, you will not be able to
access the Bal seal.

Chapter 4: Maintenance 81

3 Move the aspirator arm to the left and hold it.

aspirator arm bar

aspirator arm

4 Remove the retainer from the SIT by turning it in the direction shown.

82 BD FACSCanto Flow Cytometer Reference Manual

5 Remove the Bal seal by pulling it off with your fingers.

Bal seal

6 Install the new Bal seal

spring-side up.

Chapter 4: Maintenance 83

7 Rinse the retainer in DI water to remove saline deposits before replacing it

on the SIT.

8 Reinstall the retainer over the SIT and tighten in the direction shown.

9 Test by manually loading a tube onto the SIT and running fluid.

Resetting the Cytometer Circuit Breaker

1 Toggle the cytometer circuit breaker switch to on.

cytometer circuit breaker, on

2 If it is off, turn on the cytometer main power.

84 BD FACSCanto Flow Cytometer Reference Manual

main power
button

Reconnecting the Ethernet and RS232 Cables

The cytometer connects to and communicates with the workstation through
Ethernet and RS232 network cables. If these cables should become disconnected,
use the following diagram to reconnect them.

Figure 4-6 Ethernet and RS232 network cable

RS232 cable

Ethernet cable

Both cables connect to ports on the PC workstation. As the make and model of
the workstation might vary, refer to the documentation that came with your
system for information.

Reconnecting the Fluidics Cart Tubing

Should any plugs, cords, or tubings become accidentally disconnected, use the
following diagrams to reconnect the fluidics cart to the cytometer (Figure 4-7 on
page 86).

; Tip The ports and connectors are color coded.

Chapter 4: Maintenance 85

Figure 4-7 Cytometer and fluidics cart ports

a

b

c

d

g

cytometer ports fluidics cart ports

e

f

h

i

j

k

Cytometer ports and their corresponding fluidics cart ports are listed in
Table 4-1. For example, ensure the tubing for f. Sheath (B) connects to the port
labeled i. Fluid Out. Table 4-2 on page 87 lists port functions.

Table 4-1 Correspondence of cytometer ports to fluidics cart ports

Port or Button on Cytometer Port on Fluidics Cart

a. System Power —

l

m

n

o

b. Power Out k. Power In

c. Air In m. Air Out

d. Was t e (A) o. Was t e

e. Communications j. Communication

f. Sheath (B) i. Fluid Out

g. Wa s t e (A) n . Wa ste

— h. On/Off

—l. Air In

86 BD FACSCanto Flow Cytometer Reference Manual

Do not plug the fluidics cart power cord into a wall outlet. Plug the cord
into the cytometer only. This ensures proper electrical grounding and
protects against electrical shock or damage to the cytometer.

Table 4-2 Function of ports, buttons, and switches

Port or Switch Additional Information

a. System Power Powers both cytometer and fluidics cart

b. Power Out Connects to fluidics cart

c. Air In 70±5 psi

d. Waste (A) Waste out (aspirated)

e. Communications Data port

f. Sheath (B) BD FACSFlow solution port

g. Waste (A) Waste out (non-aspirated)

h. On/Off Auxiliary air supply switch. Keep in off

position unless connected to house air.

i. Fluid Out BD FACSFlow solution port

j. Communication Data port

k. Power In Connects to cytometer. Do not connect to

wall outlet

l. Auxiliary Air In There will be no tubing on this port unless

connected to house air.

m. Air Out Sends compressed air to cytometer

n. Waste Waste in (non-aspirated)

o. Waste Waste in (aspirated)

Chapter 4: Maintenance 87

Replacing the Fluidics Level Sensors

Replace the fluidics level sensors when instructed to do so by a BD Biosciences
service representative. Before you replace a sensor, try rinsing it with DI water.

If you are changing the sensor on the waste tank, use proper precaution and
wear suitable protective clothing, eyewear, and gloves. All biological
specimens, and materials coming into contact with them, can transmit
potentially fatal disease.

The waste tank can become pressurized when the cytometer is running.
Always disconnect the tank from the fluidics cart and wait at least 30
seconds for pressure to dissipate before you remove the level sensor cap.

Materials

• replacement fluidics sensor probe—see Other Replacement Parts on
page 129 for ordering

• proper protective equipment

88 BD FACSCanto Flow Cytometer Reference Manual

Procedure

1 Ensure that the cytometer is not acquiring events.

2 Detach the sensor and fluid line from the cart.

3 Unscrew the cap on the cubitainer.

4 Remove the level sensor assembly and discard into a suitable receptacle.

Chapter 4: Maintenance 89

5 Put a new level sensor assembly on the cubitainer or tank. Hand-tighten the

cap until it is fully closed.

6 Ensure use of the correct assembly (Figure 4-8).

Figure 4-8 Level sensor assemblies

waste level sensor
(red connector)

BD FACSFlow level sensor
(blue connector)

7 Reattach the sensor line and fluidics lines.

To ensure that the appropriate solutions are dispensed, make sure the label
on the container matches the labeled port on the fluidics cart.

8 If you replaced any level sensors (other than the waste sensor), prime the

affected fluidics lines.

Important: Continue with the procedure in Priming Fluidics Lines on
page 71.

90 BD FACSCanto Flow Cytometer Reference Manual

BD FACSClean solution and
BD FACS shutdown solution
level sensor (green connector)

Replacing the Fluidics Cart Fuses

Power surges and other electrical events could cause a fuse to blow. Use the
following procedure to replace the fuses.

Materials

• two replacement fuses (see Installation Kit on page 128 for ordering)

• small screwdriver

Removing the Old Fuses

To protect against shock, always turn off the cytometer and unplug the
power cord before performing this procedure.

1 Shut down the cytometer and turn off the cytometer power.

2 Unplug the power cord from the wall outlet and the fluidics cart.

Removing the plug allows easier access to the fuse door.

3 Insert a small screwdriver into the slot and gently pry the fuse door open.

slot for screwdriver

fuse access door

circuit breaker

empty socket,
plug removed

Chapter 4: Maintenance 91

4 Gently pry out the fuse drawer until you can grip it.

fuse drawer

fuse door

5 Remove the fuse drawer, and remove and dispose of the old fuses.

; Tip Note the positions of the old fuses before you remove them. Duplicate

these positions with the new fuses.

6 Replace both fuses.

Make sure the fuse sits as shown:

right wrong

92 BD FACSCanto Flow Cytometer Reference Manual

For protection against risk of fire, replace fuses only with those provided by
BD Biosciences.

7 Make sure the text for your area’s voltage is right-side up.

8 Slide the drawer back into the cytometer until it snaps into place.

9 Close the fuse access door.

10 Reconnect the power cord to the fluidics cart.

11 Plug the cytometer power cord into the wall outlet and switch on the power.

Chapter 4: Maintenance 93

94 BD FACSCanto Flow Cytometer Reference Manual

5

Troubleshooting

The tips in this section can help you troubleshoot issues that might arise when
using this cytometer. For software troubleshooting, refer to the BD FACSCanto

Clinical Software Reference Manual, the BD FACSDiva Software Reference
Manual, or the BD FACSCanto Instructions for Use.

If you need additional assistance, contact BD Biosciences. Refer to our website,
bdbiosciences.com, for up-to-date contact information.

Troubleshooting suggestions can be found under the following topics:

• Cytometer Troubleshooting on page 96

• Fluidics Cart Troubleshooting on page 101

• Loader Troubleshooting on page 102

95

Cytometer Troubleshooting

Observation Possible Causes Recommended Solutions

Flow cell will not fill Air in bubble filter Run the Bubble Filter Purge

command at least once (you might
need to run it several times).
When finished, run De-gas Flow
Cell. See Purging the Bubble Filter
on page 78.

Fluidics cart power off Turn on the power to the fluidics

cart by resetting the fluidics cart
circuit breaker switch (Figure 1-7
on page 27). Always use the
cytometer power button, located
on the left side of the cytometer, to
turn the system off and on.

No pressure in the plenum • Check air tubing connections.

• Check air tubing for kinks.

Sheath line disconnected

1 Check cubitainer-to-fluidics

2 Check tubing for kinks.

BD FACSFlow cubitainer
empty

Air in BD FACSFlow filter
(fluidics cart)

Air lock in BD FACSFlow
filter (on fluidics cart)

Replace the BD FACSFlow
cubitainer. See Changing a
Cubitainer on page 68.

Purge air from the filter. See
Purging the Fluidics Filters on
page 57.

See Removing an Air Lock on
page 72.

96 BD FACSCanto Flow Cytometer Reference Manual

cart, and fluidics cart–to­cytometer connections. See
Reconnecting the Fluidics Cart
Tubing on page 85.

Cytometer Troubleshooting (continued)

Observation Possible Causes Recommended Solutions

Flow cell will not fill
(continued)

Fluidics pressure errors Air lock in filter • Check the filter in the fluidics

Fluid backfill into
sample tube

No air pressure in cart • If the cart is attached to an

auxiliary air supply, switch on
auxiliary air.

• If the cart is not attached to an
auxiliary air supply, switch off
auxiliary air.

cart. Verify the bottom bleeder
valve on the filter is fully
tightened. Open the top bleeder
valve. If no fluid leaks out,
remove the air lock as
described in Removing an Air
Lock on page 72.

Cracked tube • Transfer sample to new tube.

• Make sure you are using
appropriate tubes. See System
Requirements on page 31.

Bal seal improperly installed
or worn

Air lock in BD FACSFlow
filter

Reinstall or replace the Bal seal.
See Replacing the Bal Seal on
page 80.

See Removing an Air Lock on
page 72.

Liquid leakage around
cytometer base

QC fails after Long
Clean

Interior valve failure

Residual BD FACSClean
solution in lines

1 Turn off the cytometer power.

2 Clean up the liquid, using

proper precautions.

3 Call BD Biosciences.

Run Fluidics Startup to flush the
system with sheath fluid. Repeat
until QC passes.

Chapter 5: Troubleshooting 97

Cytometer Troubleshooting (continued)

Observation Possible Causes Recommended Solutions

Cytometer on, but no
response to software
commands

Software does not
connect to cytometer

Bad keyboard or mouse
connection

Communication failure
(Ethernet error) between
computer and cytometer

Check keyboard or mouse
connections to computer. Refer to
the documentation that came with
your workstation.

1 Turn off the computer and the

cytometer.

2 Reseat the Ethernet cable,

located on the right side of the
flow cytometer. See Figure 4-6
on page 85.

3 Turn on the cytometer, then the

computer.

4 Using hyperterminal, type

reboot. Verify that VxWorks
launches and no errors are
reported.

BD FACSDiva software
running at same time as
BD FACSCanto clinical
software

Cytometer power is off Turn power on.

Internal firmware error Cycle power on cytometer.

Quit the software you are not
using or put the software in
Standby.

Cytometer and fluidics
cart will not turn on

Power cords disconnected
from wall socket or cytometer

Cytometer circuit breaker
tripped

Blown fuse in cytometer
controller

Reconnect the power cord to the
wall socket or the cytometer.

Switch on the cytometer circuit
breaker, followed by the
cytometer power (if off).

Turn off the cytometer main
power and call BD Biosciences.

98 BD FACSCanto Flow Cytometer Reference Manual

Cytometer Troubleshooting (continued)

Observation Possible Causes Recommended Solutions

Tube does not load, or
sample tube does not fit
snugly on SIT

Higher than expected
CVs for some (brightest)
populations during DNA
analysis

Improper sample tube used Use Falcon brand 12 x 75-mm

polystyrene test tubes,
BD Trucount tubes, or BD FACS
7-color setup bead tubes. See
System Requirements on page 31.

Bal seal improperly installed
or worn

Defective or cracked tube • Transfer sample to new tube.

Bal seal improperly installed
or worn

Adapter lever in incorrect
position for manual loading

Possible pre-amp non­linearity with very bright
signals

Reinstall or replace the Bal seal.
See Replacing the Bal Seal on
page 80.

• Make sure you are using
appropriate tubes. See System
Requirements on page 31.

Reinstall or replace the Bal seal.
See Replacing the Bal Seal on
page 80.

Make sure the lever is in the
manual loading position. Modify
the cytometer appropriately. See
Changing to Manual Loading on
page 39.

Set G
at channel 25,000 or less.

diploid population peak

0/G1

>20% difference
between Measured and
Reference laser power

BD FACSDiva software
not launching

Flow cell access door open Close the door completely.

Cytometer.dat file not found • Install saved cytometer.dat file,

or

• call BD Biosciences for
assistance.

Chapter 5: Troubleshooting 99

Cytometer Troubleshooting (continued)

Observation Possible Causes Recommended Solutions

Fluid leaking from SIT or
aspirator arm

Remove tube sequence not
followed correctly, cytometer
still in acquisition mode

Interior valve failure or leak

Waste line to fluidics cart
disconnected

Perform the tube removal
sequence again, even if a tube is
no longer on the SIT.

1 Turn off the cytometer power.

2 Clean up the liquid, using

proper precautions.

3 Call BD Biosciences.

1 Turn off the cytometer power.

2 Clean up the liquid, using

proper precautions.

3 Check that waste lines are

securely plugged in.

4 Turn on the cytometer power.

100 BD FACSCanto Flow Cytometer Reference Manual