BD LSRFortessa User manual

bdbiosciences.com
23-11093-00 Rev. A
3/2010
For Research Use Only
Becton, Dickinson and Company
BD Biosciences
San Jose, CA 95131
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Fax [phone]
[email protected]
BD Biosciences
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BD LSRFortessa
Cell Analyzer
User’s Guide

Copyrights
© 2010, 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.
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Patents
The BD LSRFortessa™ Cell Analyzer is covered by one or more of the following US Patents and foreign
equivalents: 7,129,505; 6,944,338; 6,809,804; 6,683,314; 6,014,904
APC-Cy7: US 5,714,386
BD FACS lysing solution: US 4,654,312; 4,902,613; 5,098,849
Regulatory information
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Class I (1) Laser Product

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, 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 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.
Compliance information
NOTICE: This laboratory equipment has been tested and found to comply with the EMC and the Low
Voltage Directives. This includes FCC, Part 15 compliance for a Class A Digital Device.
CAUTION: Any unauthorized modifications to this laboratory equipment may affect the Regulatory
Compliance items stated above.
History
Revision Date Change made
647247 Rev. A 5/2009 New document
23-11093-00 Rev. A 3/2010 Updated

Contents
Chapter 1: About this guide 1
What this guide covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Conventions used in this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
About the BD LSRFortessa documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Technical assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Chapter 2: Introduction 9
Instrument overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Sheath and waste containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Chapter 3: Cytometer setup 23
Starting the cytometer and computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Preparing the sheath container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Removing air bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Preparing the waste container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Priming the fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
About the optical filters and mirrors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Changing optical filters and mirrors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Custom configurations and baselines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

BD LSRFortessa Cell Analyzer User’s Guidevi
Chapter 4: Maintenance 39
Maintenance overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Cleaning the fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Shutting down the cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Flushing the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Maintaining the waste management system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Replacing the waste air filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Changing the sheath filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Changing the Bal seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Changing the sample tube O-ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Chapter 5: Optimizing cytometer settings 57
Cytometer settings workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Verifying the configuration and user preferences . . . . . . . . . . . . . . . . . . . . . . . . . 61
Running a performance check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Setting up an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Creating application settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Recording compensation controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Calculating compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Chapter 6: Recording and analyzing data 81
Data recording and analysis workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Preparing the workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Recording data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Analyzing data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Reusing an analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Chapter 7: Technical overview 97
About fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
About optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
About electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Contents vii
Chapter 8: Troubleshooting 115
Cytometer troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Electronics troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Chapter 9: Detector array configurations 129
Fluorescence spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
About configuration maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
About the base configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Base configuration octagon and trigon maps . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Special Order configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Chapter 10: Manual settings 163
About laser delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Optimizing laser delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Adjusting area scaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Chapter 11: Supplies and consumables 175
Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Index 181

1
About this guide
This section includes these topics:
zWhat this guide covers (page 2)
zConventions used in this guide (page 3)
zAbout the BD LSRFortessa documentation (page 5)
zTechnical assistance (page 7)

BD LSRFortessa Cell Analyzer User’s Guide2
What this guide covers
This guide describes the procedures necessary to operate and
maintain your BD LSRFortessa™ cell analyzer. Because
many cytometer functions are controlled by BD FACSDiva™
software, this guide also contains information about
software features required for basic cytometer setup and
operation.
This guide assumes you have a working knowledge of basic
Microsoft® Windows® operation. If you are not familiar
with the Windows operating system, see the documentation
provided with your computer.

Chapter 1: About this guide 3
Conventions used in this guide
About this topic This topic describes the conventions used throughout this
guide.
Safety symbols These safety symbols are used in this guide and on safety
labels to alert you to potential hazards.
Symbol Meaning
Caution alert
Identifies a hazard or unsafe practice that could
result in data loss, material damage, minor injury,
severe injury, or death
Biological hazard
Electrical hazard
Laser hazard
Hot surface; High temperatures; Burn hazard

BD LSRFortessa Cell Analyzer User’s Guide4
Text and keyboard
conventions Convention Use
Notes A note describes important features or
instructions.
Bold Bold is used to indicate software elements
such as dialogs, fields, and buttons.
Italics Italics are used to highlight book titles.
Italics are also used in describing software
to indicate specific text typed into a
window or dialog.
> The arrow is used to indicate menu
selection paths. For example, “Select
File > Print” means to select the Print
option from the File menu.
Ctrl+XWhen used with key names, a plus sign (+)
means to press two keys simultaneously.
For example, Ctrl+P means to hold down
the Control key while pressing the pkey.

Chapter 1: About this guide 5
About the BD LSRFortessa documentation
About this topic This topic describes the documentation available with the
BD LSRFortessa flow cytometer.
Publication formats This guide is provided in PDF format to provide an
eco-friendly option. All content is also included in the
BD FACSDiva software Help.
Online help The online help installed with BD FACSDiva software
includes all content from this guide and the documents listed
below. Access BD LSRFortessa online help from the Help
menu in BD FACSDiva software.
The online help is compiled from the following documents:
zBD FACSDiva Software Reference Manual: Includes
instructions or descriptions for installation and setup,
workspace components, acquisition controls, analysis
tools, and data management. Access this manual from
the BD FACSDiva Software Help menu (Help >
Literature > Reference Manual), or by double-clicking
the shortcut on the desktop.
zBD Cytometer Setup and Tracking Application Guide:
Describes how to use the Cytometer Setup and Tracking
features in BD FACSDiva software.
zBD LSRFortessa Cell Analyzer Site Preparation Guide:
Contains specifications for:
– Cytometer weight and size
– Temperature and other environmental requirements
– Electrical requirements

BD LSRFortessa Cell Analyzer User’s Guide6
zBD High Throughput Sampler User’s Guide: Describes
how to set up and operate the BD™ High Throughput
Sampler (HTS) option. It also contains a description of
BD FACSDiva software features specific to the HTS.
zBD FACSFlow Supply System User’s Guide: Describes
the optional automated sheath and waste fluid control
system designed for use with the BD™ LSR II and
BD LSRFortessa.

Chapter 1: About this guide 7
Technical assistance
About this topic This topic describes how to get technical assistance.
Getting assistance from
this guide
For technical questions or assistance in solving a problem:
zRead the sections of the documentation specific to the
operation you are performing. See About the
BD LSRFortessa documentation (page 5) for more
information.
zSee Troubleshooting (page 115).
Contacting technical
support
If additional assistance is required, contact your local
BD Biosciences customer support representative or supplier.
When contacting BD Biosciences, have the following
information available:
zProduct name, part number, and serial number
zVersion of BD FACSDiva software you are using
zAny error messages
zDetails of recent system performance
For cytometer support from within the US, call
877.232.8995.
For support from within Canada, call 888.259.0187.
Customers outside the US and Canada, contact your local
BD representative or distributor.

Need more information fast?
Information about this product is also available in your
software's Help system.
You can keep the Help window open while you use the
software or print the information directly from the window.
Internet access is not required to use the Help system.

2
Introduction
The following topics are covered in this section:
zInstrument overview (page 10)
zComponents (page 11)
zFluidics (page 13)
zSheath and waste containers (page 17)
zOptics (page 18)
zWorkstation (page 21)

BD LSRFortessa Cell Analyzer User’s Guide10
Instrument overview
The BD LSRFortessa is an air-cooled multi-laser benchtop
flow cytometer with the ability to acquire parameters for a
large number of colors. It uses fixed-alignment lasers that
transmit light through a flow cell to configurable octagon
and trigon detector arrays. These detectors collect and
translate the resulting fluorescence signals into electronic
signals. Cytometer electronics convert these signals into
digital data.

Chapter 2: Introduction 11
Components
About this topic This topic describes the instrument’s components.
Instrument overview
fluidics
interconnects
Control panel
Sample injection port (SIP)
Side drawer (octagon or trigon
detector arrays)
Power switch (right side)
Heat ventilation slots
Front doors (trigon detector
arrays)
Caution! Do not place any objects on top of the instrument.
Blocking the ventilation may cause the instrument to
overheat.
Caution: Electrical Hazard! Do not place liquids on top of the
instrument. Any spill of liquid into the ventilation openings
could cause electrical shock or damage to the instrument.

BD LSRFortessa Cell Analyzer User’s Guide12
Power switch The power switch is located on the right side of the
instrument.
Control panel The control panel contains the following fluidics controls:
zSample flow rate control buttons
zFluid control buttons
zSample fine adjust knob
Related topics zFluidics (page 13)
zOptics (page 18)
Sample flow rate control buttons Fluid control buttons