BD LSRFortessa™ X-20
Cell Analyzer
User’s Guide

For Research Use Only
23-11947-01
10/2020

Becton, Dickinson and Company
BD Biosciences
2350 Qume Drive
San Jose, CA 95131 USA
bdbiosciences.com
ResearchApplications@bd.com

BD Biosciences
European Customer Support
Tel +32.53.720.600
help.biosciences@bd.com

Copyrights© 2020, 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, inany 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 guidehas 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.

Trademarks
BD, the BD Logo, FACS, FACSDiva, FACSFlow, and FACSClean are trademarks of Becton, Dickinson andCompany or its affiliates. All other trademarks are the property of their respective owners.© 2020 BD. All
rights reserved.
Alexa Fluor™ and Pacific Blue™ are trademarks of Life Technologies Corporation.
Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare andCarnegie Mellon University and are made and sold under license from GE Healthcare only for research and invitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial
Avenue, Piscataway, NJ 08855-1327, USA.

Patents
APC-Cy7: US 5,714,386

Regulatory information
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Class 1 Laser ProductFCC information
WARNING: Changes or modifications to this unit not expressly approved by the party responsible forcompliance 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 theinterference 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 InterferenceCausing Equipment Regulations. Cet appareil numérique de la classe A respecte toutes les exigences duRéglement 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 made23-11947-00

4/2013

Initial release23-11947-01

10/2020

Removed references of FACSRinse and added BD Detergent
Solution Concentrate in place of FACSRinse.

Contents
Chapter 1: About this guide9What this guide covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
About the BD LSRFortessa X-20 documentation . . . . . . . . . . . . . . . . . . . . . . . . 11
Instrument technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Chapter 2: Introduction

15

Instrument overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Sheath and waste containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Chapter 3: Cytometer setup

29

Starting the cytometer and computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Preparing the sheath container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Removing air bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Preparing the waste container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Priming the fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
About the optical filters and mirrors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Changing optical filters and mirrors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Custom configurations and baselines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

6

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Chapter 4: Maintenance

45

Maintenance overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Cleaning the fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Shutting down the cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Flushing the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Maintaining the waste management system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Replacing the waste air filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Changing the sheath filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Changing the Bal seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Changing the sample tube O-ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Chapter 5: Optimizing cytometer settings

63

Cytometer settings workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Verifying the configuration and user preferences . . . . . . . . . . . . . . . . . . . . . . . . . 66
Running a performance check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Setting up an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Creating application settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Recording compensation controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Calculating compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Chapter 6: Recording and analyzing data

85

Data recording and analysis workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Preparing the workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Recording data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Analyzing data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Reusing an analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Chapter 7: Technical overview

99

About fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
About optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
About electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Contents

Chapter 8: Troubleshooting

117

Cytometer troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Electronics troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Chapter 9: Detector array configurations

129

Fluorescence spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
About configuration maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
About the base configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Base configuration polygon maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Special order configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Chapter 10: Manual settings

165

About laser delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Optimizing laser delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Adjusting area scaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Chapter 11: Supplies and consumables

175

Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Index

179

7

1
About this guideThis chapter covers the following topics:•

What this guide covers (page 10)•

Conventions (page 11)•

About the BD LSRFortessa X-20 documentation (page 11)•

Instrument technical support (page 13)

10

BD LSRFortessa X-20 Cell Analyzer User’s Guide

What this guide covers
This guide describes the procedures necessary to operate andmaintain your special order BD LSRFortessa™ X-20 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 andoperation.
This guide assumes you have a working knowledge of basic
Microsoft® Windows® operation. If you are not familiar with theWindows operating system, see the documentation provided withyour computer.

Chapter 1: About this guide

Conventions
Introduction

The following table lists the safety symbols used in this guide toalert you to potential hazards.

Safety symbols
SymbolMeaning
Caution alert
Identifies a hazard or unsafe practice that could result indata loss, material damage, minor injury, severe injury, or
death
Biological hazard

Electrical hazard

Laser hazard

About the BD LSRFortessa X-20 documentation
Introduction

This topic describes the documentation available with the special
order BD LSRFortessa X-20 cell analyzer.

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.

11

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Help system

The help system installed with BD FACSDiva software includes allcontent from this guide and the documents listed below. Access the
BD LSRFortessa X-20 help system from the Help menu in
BD FACSDiva software. Internet access is not required to use thehelp system.
The help system is compiled from the following documents:•

BD 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 theBD FACSDiva Software Help menu (Help > Documentation >Reference Manual), or by double-clicking the shortcut on thedesktop.

•BD Cytometer Setup and Tracking Application Guide:
Describes how to use the BD® Cytometer Setup and Tracking(CS&T) features in BD FACSDiva software.

•

BD LSRFortessa X-20 Cell Analyzer Site Preparation Guide:
Contains specifications for:– Cytometer weight and size
– Temperature and other environmental requirements– Electrical requirements•

BD 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.

•BD FACSFlow Supply System User’s Guide: Describes theoptional automated sheath and waste fluid control systemdesigned for use with the BD® LSR II, BD LSRFortessa™, andBD LSRFortessa X-20.

Chapter 1: About this guide

Instrument technical support
Introduction

This topic describes how to get technical assistance.

Contacting
technical support

If technical assistance is required, contact your local
BD Biosciences customer support representative or supplier.
When contacting BD Biosciences, have the following information
available:•

Product name, part number, and serial number•

Version of BD FACSDiva software you are using•

Any error messages•

Details of recent system performance

To contact customer support:
1. Go to bdbiosciences.com.
2. Select your region. You see information in your local language.
3. Click Go.
4. Click the Support link for details for your local region.

13

This page intentionally left blank2
Introduction
This chapter covers the following topics:•

Instrument overview (page 16)•

Components (page 17)•

Fluidics (page 19)•

Sheath and waste containers (page 23)•

Optics (page 23)•

Workstation (page 27)

16

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Instrument overview
The special order BD LSRFortessa X-20 cell analyzer is an aircooled multi-laser benchtop flow cytometer with the ability toacquire parameters for a large number of colors. It uses
fixed-alignment lasers that transmit light through a flow cell to
configurable polygon detector arrays. These detectors collect andtranslate the resulting fluorescence signals into electronic signals.
Cytometer electronics convert these signals into digital data.

Chapter 2: Introduction

Components
Introduction

This topic describes the instrument’s components.

Instrument
overview
Heat ventilation slots

Power switch (right side)

Sample injection port (SIP)

Side drawer (polygon
detector arrays)

Control panel

Caution! Do not place any objects on top of the
instrument. Blocking the ventilation may cause theinstrument to overheat.

17

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

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.
Power switch

The power switch is located on the right side of the instrument.

Control panel

The control panel contains the following fluidics controls:•

Sample flow rate control buttons•

Fluid control buttons•

Sample fine adjust knob

The control panel contains the following system indicators:

SAMPLEFINE ADJ•

Acquisition status•

System power

RUN

STANDBY

PRIME

LOW

MED

HIGH

ACQUISITION STATUS

SYSTEM POWER

Fluid control buttons

More information

Sample flow rate control buttons•

Fluidics (page 19)•

Optics (page 23)

System indicators

Chapter 2: Introduction

Fluidics
Introduction

This topic describes the fluidics system.

PurposeThe purpose of the fluidics system is to carry the sample out of thesample tube and into the sensing region of the flow cell. Cells arecarried in the sample core stream in single file and measured
individually.

Sample flow rate
control

Three flow rate control buttons (LOW, MED, and HIGH) set the
sample flow rate through the flow cell. The SAMPLE FINE ADJ
knob allows you to adjust the rate to intermediate levels.
When the SAMPLE FINE ADJ knob is at its midpoint, the sample
flow rates at the LOW, MED, and HIGH settings areapproximately 12, 35, and 60 µL/min of sample, respectively. The
knob turns five full revolutions in either direction from its
midpoint, providing sample flow rates from 0.5–2X the midpointvalue. For example, if the LOW button is pressed, the knob will
give flow rates from approximately 6–24 µL/min.

19

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Fluid control

Three fluid control buttons (RUN, STANDBY, and PRIME) set thecytometer mode.
•

RUN. Pressurizes the sample tube to transport the sample
through the sample injection tube and into the flow cell.
The RUN button is green when the sample tube is on and the
support arm is centered. When the tube support arm is moved
left or right to remove a sample tube, the cytometer switches toan automatic standby status to conserve sheath fluid, and the
RUN button changes to orange.

•

STANDBY. Stops fluid flow to conserve sheath fluid.
When you leave the cytometer for more than a few minutes,
place a tube containing 1 mL of deionized (DI) water on thesample injection port (SIP) and press STANDBY.

•PRIME. Prepares the fluidics system by draining and filling theflow cell with sheath fluid.
The fluid flow initially stops and pressure is reversed to force
fluid out of the flow cell and into the waste container. After apreset time, the flow cell fills with sheath fluid at a controlledrate to prevent bubble formation or entrapment. At
completion, the cytometer switches to standby mode.

System indicators

The two system indicators (ACQUISITION STATUS and SYSTEM
POWER) show the current status of the cytometer.
•

ACQUISITION STATUS. The light is green when the sample
is being acquired by the cytometer.

•

SYSTEM POWER. The light is blue when the cytometer
power is on.

Chapter 2: Introduction

Sample injection
port

The SIP is where the sample tube is installed. The SIP includes the
sample injection tube and the tube support arm. Samples are
introduced through a stainless steel injection tube equipped withan outer droplet containment sleeve. The sleeve works inconjunction with a vacuum pump to eliminate droplet formationof sheath fluid as it backflushes from the sample injection tube.

Outer sleeve
Sample injection tube

Tube support arm

Sample injection tube. Stainless steel tube that carries sample fromthe sample tube to the flow cell. This tube is covered with an outersleeve that serves as part of the droplet containment system.
Tube support arm. Arm that supports the sample tube andactivates the droplet containment system vacuum. The vacuum ison when the arm is positioned to the side and off when the arm iscentered.
Note: If a sample tube is left on the SIP with the tube support arm
to the side (vacuum on), the sample will be aspirated into the waste
container.

21

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Cautions whenusing the HTS
optionCaution: Biohazard! When using the BD LSRFortessa
X-20 cell analyzer with the HTS, ensure that the HTS iscompletely pushed into the operating position before
removing the droplet containment module (DCM) sleeveor disconnecting the sample coupler from the SIP. This is
to avoid accidental leakage of potentially biohazardous
liquids directly onto the instrument. With the HTS in theproper location, the containment dish with padding isdirectly below the SIP.
Caution! If you are using the HTS option, always slidethe HTS mount slowly to prevent sample crosscontamination when the wells are full. Never move the
HTS when it is in operation.
Caution! Do not lean on or put any weight on the HTS as
it could damage the instrument.

Dropletcontainment
moduleThe DCM prevents sheath fluid from dripping from the SIP andprovides biohazard protection.
When no sample tube is installed on the SIP, sheath fluid
backflushes through the sample injection tube. This backflush
helps prevent carryover of cells between samples. The DCM
vacuum is activated when the sample tube is removed and the tube
support arm is moved to the side. Sheath fluid is aspirated as it
backflushes the sample injection tube.

Chapter 2: Introduction

Sheath and waste containers
Introduction

This topic describes the sheath and waste containers. The sheath
and waste containers are outside the cytometer and are positionedon the floor.
Note: If your system is using the BD FACSFlow™ supply system,
please see the documentation provided with your system.

Sheath container

The sheath container has a capacity of 8 L. Sheath fluid is filteredthrough an in-line, interchangeable filter that prevents small
particles from entering the sheath fluid lines.
Caution! Do not fill the sheath tank to its maximum
capacity (8 L). When an overfull tank is pressurized,
erratic cytometer performance can result.

Waste container

The waste container has a capacity of 10 L. An alarm sounds when
the container becomes full.

More information•

Preparing the sheath container (page 31)•

Preparing the waste container (page 36)

OpticsIntroduction

This topic describes the optical components for the special order
BD LSRFortessa X-20 cell analyzer including:•Detector arrays•

Laser options•

Optical filters•

Signal detectors

23

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Detector arrays

The BD LSRFortessa X-20 detector arrays consist of polygons.
Each polygon can be outfitted with two to eight PMTs and can
detect up to eight signals.

Locked cover (lasers
accessible by BD Service only)

Side drawer with polygons

Laser options

The special order BD LSRFortessa X-20 cell analyzer can be
configured with up to five lasers as listed in the following table.
The cytometer can also be configured with up to five lasers from avariety of wavelengths and powers through our special orderresearch program.
Laser

Wavelength (nm)Power (mW)

Blue

488

50

Red

640

40

Violet

405

50

NUV

375

50

Yellow-green

561

50

Chapter 2: Introduction

Optical filters

Optical filters attenuate light or help direct it to the appropriate
detectors. The name and spectral characteristics of each filter
appear on its holder.
There are two types of optical filters in the BD LSRFortessa X-20:•

Longpass dichroic filters (LPs). Transmit wavelengths that are
longer than the specified value and reflect all light below thespecified wavelength.

•

Bandpass filters (BPs). Pass a narrow spectral band of light.

When dichroic filters are used as steering optics to direct different
color light signals to different detectors, they are called dichroic
mirrors. LP dichroic mirrors transmit longer wavelengths to one
detector while reflecting shorter wavelengths to a different
detector.
The BD LSRFortessa X-20 cell analyzer polygon detector arrays
use dichroic longpass mirrors on the inside, and bandpass filters on
the outside of the filter holders. You can customize the arrays withother wavelengths of filters and mirrors.

Bandpass filters
Longpass dichroic mirrors

25

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Signal detectors

Light signals are generated as particles pass through the laser beamin a fluid stream. When these optical signals reach a detector,
electrical pulses are created that are then processed by theelectronics system.
There are two types of signal detectors in the BD LSRFortessa
X-20 cell analyzer:

More information•

Photomultiplier tubes (PMTs). Used to detect the weakersignals generated by side scatter and all fluorescence channels.
These signals are amplified by applying a voltage to the PMTs.

•

Photodiodes. Less sensitive to light signals than the PMTs. A
photodiode is used to detect the stronger forward scatter (FSC)
signal. However, an optional PMT for detecting FSC isavailable through the BD special order research program.

•

Optical filter theory (page 104)•

About the base configuration (page 133)•

Special order configurations (page 150)

Chapter 2: Introduction

Workstation
Introduction

This topic describes the components of the BD LSRFortessa X-20
workstation.

Workstation
components

Acquisition, analysis, and most instrument functions are controlled
by the BD LSRFortessa X-20 workstation. It includes a PC, one or
two monitors, and a printer.
Your workstation is equipped with the following:More information•

Microsoft Windows operating system•

BD FACSDiva software version 7.0 or later for dataacquisition and analysis•

Software documentation including the help system•

About the BD LSRFortessa X-20 documentation (page 11)27

This page intentionally left blank

3
Cytometer setup
This chapter covers the following topics:•

Starting the cytometer and computer (page 30)•

Preparing the sheath container (page 31)•

Removing air bubbles (page 33)•

Preparing the waste container (page 36)•

Priming the fluidics (page 39)•

About the optical filters and mirrors (page 40)•

Changing optical filters and mirrors (page 42)•

Custom configurations and baselines (page 43)

30

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Starting the cytometer and computer
Introduction

This topic describes how to start the cytometer and turn on thecomputer.
Note: If your system is using the BD FACSFlow supply system,
make sure that the BD FACSFlow supply system is powered on
before the cytometer.

Procedure

To start the cytometer:
1. Turn on the power to the flow cytometer.
2. Allow 30 minutes for the optical system temperature tostabilize.
Caution! Failure to warm up and stabilize theinstrument could affect sample data.
3. Turn on the computer and log in to Windows.
Note: You can turn on the power to the flow cytometer andthe workstation in any order.
4. Start BD FACSDiva software by double-clicking the shortcut
on the desktop, and log in to the software.
5. Check the Cytometer window in BD FACSDiva software toensure that the cytometer is connected to the workstation.
The cytometer connects automatically. While connecting, themessage Cytometer Connecting is displayed in the status areaof the Cytometer window. When connection completes, themessage changes to Cytometer Connected.

Chapter 3: Cytometer setup

If the message Cytometer Disconnected appears, seeElectronics troubleshooting (page 127).

Preparing the sheath container
Introduction

This topic describes how to prepare the sheath container.
Note: If your system is using the BD FACSFlow supply system,
please see the documentation provided with your system.

When to check the
sheath container

Check the fluid levels in the sheath container every time you use
the cytometer. This ensures that you do not run out of sheath fluid
during an experiment.

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Sheath container
components

Clamp knob
Cap handle

Air line (green)

Tank handle
Vent valve

Sheath fluid line (blue)
to cytometer

Filter assembly

Procedure

To prepare the sheath container:
1. Verify that the flow cytometer is in standby mode.
Press the STANDBY button on the control panel if necessary.
2. Disconnect the green air line from the sheath container.
3. Depressurize the sheath container by pulling up on the ventvalve.
4. Remove the sheath container lid.
Unscrew the clamp knob and push down to loosen, if
necessary. Tilt the cap to the side to remove it from the tank.

Chapter 3: Cytometer setup

5. Add 6 L of sheath fluid, such as BD FACSFlow solution, to thesheath container.
Caution! Do not fill the sheath tank to its maximum
capacity (8 L). When an overfull tank is pressurized,
erratic cytometer performance can result.
6. Replace the sheath container lid.
7. Reconnect the green air line.
8. Make sure the gasket on the inside lip of the sheath lid isseated correctly and has not slipped out of position.
If the gasket is not seated correctly, the tank will not pressurize
properly.
9. Close the sheath lid and tighten the clamp knob to finger-tight.
Ensure that the blue sheath fluid line is not kinked.
More information•

Removing air bubbles (page 33)•

Changing the sheath filter (page 56)

Removing air bubbles
Introduction

This topic describes how to remove trapped air bubbles in the
sheath filter and the sheath line. Air bubbles can occasionallydislodge and pass through the flow cell, resulting in inaccurate
data.

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Procedure

To remove air bubbles:
1. Check the sheath filter for trapped air bubbles.
Cytometer fluid line
(roller clamp not visible)

Vent fitting

Vent line

2. If bubbles are visible, gently tap the filter body with yourfingers to dislodge the bubbles and force them to the top.
Caution! When removing air bubbles, do not
vigorously shake, bend, or rattle the sheath filter or
you might damage it.

Chapter 3: Cytometer setup

3. Direct the vent line into a beaker and press the small button at
the end of the vent fitting against the side of the beaker until asteady stream of fluid empties from the filter.
ButtonVent fitting

4. Tilt the filter and verify that no trapped air remains in thefilter.
5. Repeat steps 3 and 4 until no air is observed in the filter.
6. Check the sheath line for air bubbles.
7. Open the roller clamp at the fluidics interconnect (if necessary)to bleed off any air in the line. Collect any excess fluid in a
waste container.
8. Close the roller clamp.

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Preparing the waste container
Introduction

This topic describes how to prepare the waste container.
Note: If your system is using the BD FACSFlow supply system,
please see the documentation provided with your system.
Caution: Biohazard! All biological specimens andmaterials coming into contact with them are consideredbiohazardous. Handle as if capable of transmitting
disease. Dispose of waste using proper precautions and inaccordance with local regulations. Never pipette bymouth. Wear suitable protective clothing, eyewear, andgloves.

When to check thewaste container

Check the fluid levels in the waste container every time you use thecytometer. This ensures that the waste container does not become
too full.

Chapter 3: Cytometer setup

Waste container
components
Waste tubing (orange) fromcytometer
Waste air vent filter

Level sensor line

BracketCautions

Caution: Biohazard! To avoid leakage of biohazardous
waste, put the cytometer in standby mode before
disconnecting the waste container.
Caution: Biohazard! The waste container contents might
be biohazardous. Treat contents with bleach (10% of
total volume).

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Caution! If the air vent filter on the top of the waste
container cap assembly becomes clogged, air cannot bevented from the container, causing it to swell underpressure. If you observe swelling of the waste container,
loosen the cap to relieve the pressure, and immediately
replace the air filter. See Replacing the waste air filter(page 55) for instructions. Removing the air filter or thewaste container cap assembly without first relieving thepressure might generate an unnoticeable aerosol. Useappropriate precautions when troubleshooting a clogged
air filter. Wear suitable protective clothing, eyewear, andgloves.
Procedure

To prepare the waste container:
1. Verify that the flow cytometer is in standby mode.
Press the STANDBY button on the control panel if necessary.
2. Disconnect the orange waste tubing and the black level sensor
line from the waste container.
Keep the lid on the waste container until you are ready toempty it.
3. Empty the waste container.
Caution! The waste container is heavy when full.
When emptying it, use good body mechanics toprevent injury.
4. Add approximately 1 L of bleach to the waste container andclose it.
5. Reconnect the orange waste tubing and make sure it is notkinked.
6. Reconnect the level sensor line.

Chapter 3: Cytometer setup

Priming the fluidics
Introduction

This topic describes how to prime the fluidics system.

When to prime thefluidics

Sometimes, air bubbles and debris may become lodged in the flow
cell. This is indicated by excessive noise in the forward and side
scatter parameters (FSC and SSC, respectively). In these cases, it isnecessary to prime the fluidics system.

Procedure

To prime the fluidics:
1. Move the tube support arm to the side.
2. Remove the tube from the SIP.
3. Press the PRIME fluid control button to force the fluid out of
the flow cell and into the waste container.
Once drained, the flow cell automatically fills with sheath fluid
at a controlled rate to prevent bubble formation or
entrapment. The STANDBY button turns amber after
completion.
4. Repeat the priming procedure, if necessary.
5. Install a 12 x 75-mm tube with 1 mL of DI water on the SIP
and place the support arm under the tube. Leave the cytometerin standby mode.

More information•

Cytometer troubleshooting (page 118)

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About the optical filters and mirrors
Introduction

This topic provides a description of the optical filters and mirrors.

Filter and mirror
configurations

Each PMT has an optic holder in front of it. The optic holders arelabeled with numbers indicating the wavelengths of the bandpass
filter and longpass dichroic mirror they contain (for example, 695/
40 and 685 LP, respectively). The optic holder in front of the last
PMT in the detector array contains only a bandpass filter and ismarked accordingly.

PMT A

Optic holder
handleOptic holders

The filters steer progressively shorter wavelengths of light to thenext PMT in the array as indicated by the lines and arrows on the
top of the polygon.

Chapter 3: Cytometer setup

Optic holders,filters, and mirrors

Optic holders house filters and mirrors. Your cytometer includes
several blank (empty) optic holders.
Caution! To ensure data integrity, do not leave any slots
empty in a detector array when you are using theassociated laser. Always use a blank optic holder.

Base configurations Each BD LSRFortessa X-20 cell analyzer has a base cytometer
configuration that corresponds to the layout of the installed lasers
and optics in your cytometer. This base configuration is set by yourfield service engineer.
BD FACSDiva
cytometer
configuration

Before you acquire data using BD FACSDiva software, you mustspecify a cytometer configuration. The cytometer configuration
defines which filters and mirrors are installed at each detector.
BD FACSDiva software provides a BD base configuration for yourBD LSRFortessa X-20 cell analyzer. Select Cytometer >View Configuration to create, modify, or delete custom cytometer
configurations. (See the Cytometer and Acquisition Controls
chapter of the BD FACSDiva Software Reference Manual for
details.)

More information•

Changing optical filters and mirrors (page 42)•

About the base configuration (page 133)•

Special order configurations (page 150)

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Changing optical filters and mirrors
Introduction

This topic describes how to verify that the optical filters are in theappropriate position for your particular requirements. Before you
run samples, you must set up the optical filters.
Caution: Laser Hazard! Follow the precautions outlined in
the BD LSRFortessa X-20 Safety and Limitations Guide
while changing optical filters or mirrors.

Procedure

To change a filter or mirror:
1. Access the appropriate detector array.
Polygon detector arrays are located in the left side cytometer
drawer.
2. Remove the appropriate optic holder.
3. Replace the removed optic holder with the new holder
containing the appropriate filter and mirror set.
Verify that the filters are arranged so that the longest
wavelength is in the A position and the shortest wavelength isin the last position used.
4. Close the cytometer side drawer.

More information•

Detector array configurations (page 129)

Chapter 3: Cytometer setup

Custom configurations and baselines
Introduction

This topic describes where to find information on how to create acustom configuration and define a baseline for a performancecheck.

Overview

BD Cytometer Setup and Tracking (CS&T) software is used todefine the baseline performance of your cytometer. A baseline
provides a starting point for the tracking of cytometer
performance. When running a performance check, you compare
the results to the baseline.
Some BP filters might not be normalized to CS&T settings. In thiscase, CS&T will generate sensitivity and background values (Qrand Br, respectively) that are not comparable from instrument to
instrument. They are however, still trackable on one cytometer. In
addition, you must carefully check the PMT voltages that CS&T
sets for these filters. See Optimizing cytometer settings (page 63).
Please see the latest published filter guides available on our website(bdbiosciences.com) for more information.
See the BD Cytometer Setup and Tracking Application Guide for
information on creating custom configurations and defining abaseline.

More information•

Running a performance check (page 68)

43This page intentionally left blank

4
MaintenanceThis chapter covers the following topics:•

Maintenance overview (page 46)•

Cleaning the fluidics (page 47)•

Shutting down the cytometer (page 49)•

Flushing the system (page 50)•

Maintaining the waste management system (page 53)•

Replacing the waste air filter (page 56)•

Changing the sheath filter (page 57)•

Changing the Bal seal (page 59)•

Changing the sample tube O-ring (page 61)

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Maintenance overview
Introduction

This topic provides an overview of the special order
BD LSRFortessa X-20 cell analyzer routine maintenance andcleaning procedures.

General use
guidelines

Caution: Biohazard! Coming into contact with all
biological specimens and materials is consideredbiohazardous.
Follow these guidelines whenever operating or maintaining thecytometer:•

Handle all biological specimens and materials as if they arecapable of transmitting disease.

•

Dispose of waste using proper precautions and in accordancewith local regulations.

•

Never pipette by mouth.

•

Wear suitable protective clothing, eyewear, and gloves.

For fluidics maintenance, we recommend the following cleaning
solutions: BD®
•BD® FACSClean solution•

10% bleach solution•

0.5% solution of sodium hypochlorite

Use DI water to dilute bleach and sodium hypochlorite toappropriate concentrations.
Caution! Higher concentrations of sodium hypochlorite
and use of other cleaning solutions might damage thecytometer.

Chapter 4: Maintenance

When to perform
maintenanceprocedures

Perform maintenance procedures in the following frequencies.
Frequency

Maintenance procedure

Daily



Cleaning the fluidics (page 47)

Shutting down the cytometer (page 49)

Scheduled (every
two weeks)Flushing the system (page 50)

Maintaining the waste management system(page 53)

Periodic (frequency
depends on how
often you run thecytometer)

Changing the sheath filter (page 57)

Changing the Bal seal (page 59)

Changing the sample tube O-ring (page 61)

Cleaning the fluidics
Introduction

This topic describes how to perform the daily fluidics cleaning.

Overview

Cleaning the fluidics daily prevents the sample injection tube frombecoming clogged and removes dyes that can remain in the tubing.
In addition to daily cleaning, follow this procedure immediately
after running viscous samples or nucleic acid dyes such as Hoechst,
DAPI, propidium iodide (PI), acridine orange (AO), or thiazole
orange (TO).

Procedure

To clean the fluidics:
1. Press RUN and HIGH on the cytometer fluid control panel.
2. Install a tube containing 3 mL of a cleaning solution on the SIP
with the support arm to the side (vacuum on) and let it run for
1 minute.

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For the cleaning solution, use BD FACSClean solution. See
Maintenance overview (page 46) for other recommended
cleaning solutions.
3. Move the tube support arm under the tube (vacuum off) andallow the cleaning solution to run for 5 minutes with thesample flow rate set to HIGH.
4. Repeat steps 2 and 3 with DI water.
5. Repeat steps 2 and 3 with 1.5% dilution of BD Detergent
Solution Concentrate.
Note: The BD Detergent Solution Concentrate must be diluted
before use. Mix one full 15 mL bottle of BD Detergent
Solution Concentrate into 985 mL of DI water to make 1 L
total.
Caution! Do not mix BD Detergent Solution Concentrate
and bleach because they produce chlorine gas.
6. Repeat steps 2 and 3 with DI water.
7. Press the STANDBY button on the fluidics control panel.
8. Place a tube containing no more than 1 mL of DI water on the
SIP.
A tube with 1 mL of DI water should remain on the SIP toprevent salt deposits from forming in the injection tube. This
tube also catches back drips from the flow cell.
Caution! Do not leave more than 1 mL of water on
the SIP. When the instrument is turned off or left instandby mode, a small amount of fluid will drip backinto the sample tube. If there is too much fluid in thetube, it could overflow and affect the cytometer
performance.

Chapter 4: Maintenance

Shutting down the cytometer
Introduction

This topic describes how to shut down the cytometer.

Before you begin

Each time you shut down the cytometer, perform the daily cleaning
as described in Cleaning the fluidics (page 47).

Procedure

To shut down the cytometer:
1. Place a tube of DI water on the SIP.
2. Turn off the flow cytometer.
3. Select Start > Shutdown to turn off the computer (if needed).
4. If your system is using the BD FACSFlow supply system, shut
off the BD FACSFlow supply system.
If the cytometer will not be used for a week or longer, perform asystem flush and leave the fluidics system filled with DI water toprevent saline crystals from clogging the fluidics.

More information•

Cleaning the fluidics (page 47)•

Flushing the system (page 50)

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Flushing the system
Introduction

This topic describes how to perform an overall fluidics cleaning to
remove debris and contaminants from the sheath tubing, waste
tubing, and flow cell. Perform the system flush at least every 2
weeks.
Note: If you are using the BD FACSFlow supply system, see theBD FACSFlow Supply System User’s Guide for instructions on
flushing the system.

Cautions

Procedure

Caution: Biohazard! The cytometer hardware might becontaminated with biohazardous material. Use 10%
bleach to decontaminate the instrument.
To perform a system flush:
1. Remove the sheath filter.
a. Press the quick-disconnects on both sides of the filter
assembly.
b. Remove the filter assembly.
c. Connect the two fluid lines.
Caution! Do not run detergent, bleach, or ethanol
through the sheath filter. They can break down thefilter paper within the filter body, causing particles toescape into the sheath fluid, possibly clogging theflow cell.
2. Empty the sheath container and rinse it with DI water.
3. Fill the sheath container with at least 1 L of undiluted
BD FACSClean solution.
4. Empty the waste container, if needed.
5. Open the roller clamp by the fluidics interconnect, and drain
the fluid into a beaker for 5 seconds.

Chapter 4: Maintenance

6. Remove the DI water tube from the SIP.
7. Prime the instrument twice:a. Press the PRIME button on the fluidics control panel.
b. When the STANDBY button lights (amber), press the
PRIME button again.
8. Install a tube with 3 mL of undiluted BD FACSClean solution
on the SIP and put the tube support arm underneath the tube.
See Maintenance overview (page 46) for other recommended
cleaning solutions.
9. Press RUN and HIGH on the cytometer fluid control panel.
Run for 30 minutes.
10. Press the STANDBY fluid control button and depressurize thesheath container by lifting the vent valve.
11. Empty the waste tank to avoid the mixing of Bleach and BD
Detergent Solution Concentrate.
12. Repeat steps 2 through 11 with DI water.
13. Repeat steps 2 through 11 with 1.5% dilution of BD Detergent
Solution Concentrate.
Note: The BD Detergent Solution Concentrate must be diluted
before use. Mix one full 15 mL bottle of BD Detergent
Solution Concentrate into 985 mL of DI water to make 1 L
total.
Caution! Do not mix BD Detergent Solution Concentrate
and bleach because they produce chlorine gas.
14. Repeat steps 2 through 11 with DI water.
15. Replace the sheath filter and refill the sheath container withsheath fluid.

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Next step

Test the waste management system battery as described in
Maintaining the waste management system (page 53).

Chapter 4: Maintenance

Maintaining the waste management system
Introduction

This topic describes how to maintain the waste management
system. The waste management system has an alarm powered by a
9-volt battery that you must test and change regularly to ensure
continued operation.

When to performthe battery test

Test the battery every two weeks after you flush the system.
Change the battery as needed.

Testing the battery
and alarm

To test the battery and alarm:
1. Locate the Battery Test switch on the waste container bracket.

Battery test
BATTERY TEST

2. Toggle the switch.
If the battery and the alarm are working properly, you shouldhear an alarm buzzing. If you do not hear any sound, change
the battery as described in the following section.
3. Release the switch.

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Changing thebattery

You need the following supplies to change the battery:•

Small flat-head screwdriver•

A 9-volt battery

To change the battery:
1. Insert the tip of a flat-head screwdriver into the slot and gently
slide the battery drawer out.

2. Remove the drawer.

Chapter 4: Maintenance

3. Remove the battery from the drawer.

4. Place a new 9-volt battery into the drawer.
The markings in the battery drawer show the correct battery
orientation.
5. Slide the drawer into the bracket until you feel a click.
6. Test the new battery.

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Replacing the waste air filter
Introduction

This topic describes how to replace the waste air filter. An air filter
is located on the cap assembly of the waste container.

Procedure
Caution: Biohazard! Treat a contaminated air filter asbiohazardous waste.
To replace the air filter:
1. Remove the air filter.
Hold the silicone tubing with one hand and pull off the filter
with the other hand.
2. Insert a new air filter into the silicone tubing.
3. Verify that the cap assembly on the container is tightened.

Chapter 4: Maintenance

Changing the sheath filter
Introduction

This topic describes how to change the sheath filter. The sheathfilter is connected in-line with the sheath line. It filters the sheath
fluid as it comes from the sheath container.

When to change
the sheath filter

We recommend changing the sheath filter assembly every
six months. Increased debris appearing in an FSC vs SSC plot can
indicate that the sheath filter needs to be replaced. See Supplies andconsumables (page 175) for ordering information.

Sheath filter
components

Sheath line

Vent fitting
Quick-disconnect

Vent line

Filter base

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Removing the oldfilterTo remove the old filter:
1. Place the cytometer in standby mode.
2. Remove the sheath filter assembly by pressing thequick-disconnect on both sides of the filter assembly.
3. Over a sink or beaker:• Remove the vent line from the filter and set it aside.
• Remove the filter base and set it aside.
4. Discard the used filter assembly in an appropriate receptacle.

Attaching the newfilterTo attach the new filter:
1. Connect the vent line to the new filter assembly.
Twist to attach.
2. Wrap Teflon® tape around the filter threads, then connect thefilter to the filter base.
3. Connect the sheath line to the filter assembly by squeezing thequick-disconnect.
4. Attach the cytometer fluid line to the filter assembly via thequick-disconnect.
5. Direct the vent line into a beaker and press the small button at
the end of the vent fitting against the side of the beaker until asteady stream of fluid empties from the filter.
6. Tilt the filter and verify that no trapped air remains in thefilter.
7. Repeat steps 5 and 6 as necessary to remove all trapped air.

Chapter 4: Maintenance

Changing the Bal seal
Introduction

This topic describes how to replace the Bal seal.
The sample injection tube Bal seal is a ring that forms a seal withthe sample tube and ensures proper tube pressurization.

When to change
the Bal seal

ProcedureOver time, the Bal seal becomes worn or cracked and requires
replacement. Replacement is necessary if a proper seal is notformed when a sample tube is installed on the SIP. Indications thata proper seal has not formed include:•

The tube will not stay on the SIP without the tube support
arm.

•

When the tube is installed and RUN is pressed on thecytometer, the RUN button is orange (not green).

To replace the Bal seal:
1. Remove the outer sleeve from the sample injection tube byturning the retainer counter-clockwise. Slide the outer sleeve
down and off of the sample injection tube.

Bal seal
Retainer

Outer sleeve
Sample injection tube

Work carefully. The outer sleeve can fall off as you loosen theretainer.

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2. Remove the Bal seal by gripping it between your thumb andindex finger and pulling down.

3. Install the new Bal seal spring-side up.
Ensure that the sample tube O-ring is still in place inside theretainer.
4. Re-install the retainer and outer sleeve over the sample
injection tube. Push the outer sleeve all the way up into thesample injection port and then screw the retainer into place
and tighten to finger tight. This will seat the Bal seal.
5. Install a sample tube on the SIP to ensure that the outer sleeve
has been properly installed.
If the sleeve hits the bottom of the tube, loosen the retainer
slightly and push the sleeve up as far as it will go. Tighten theretainer.

Chapter 4: Maintenance

Changing the sample tube O-ring
Introduction

This topic describes how to replace the sample tube O-ring.
The sample tube O-ring, located within the retainer, forms a sealthat allows the droplet containment vacuum to function properly.

When to replace
the O-ringCautionProcedure

Replace the O-ring when droplets form at the end of the sample
injection tube while the vacuum is operating.

Caution: Biohazard! Cytometer hardware might becontaminated with biohazardous material. Wear suitable
protective clothing, eyewear, and gloves whenevercleaning the cytometer or replacing parts.
To change the O-ring:
1. Remove the outer sleeve from the sample injection tube byturning the retainer counter-clockwise.
2. Slide the outer sleeve from the retainer.

O-ringRetainer61

3. Invert the outer droplet sleeve and allow the O-ring tofall onto the benchtop.
If the O-ring does not fall out initially, hold the O-ringwith your free hand and slide the outer sleeve to remove
the O-ring.
4. Place the new O-ring into the retainer. Make sure theO-ring is seated properly in the bottom of the retainer.
5. Replace the outer sleeve in the retainer.
6. Re-install the retainer and the outer sleeve.
7. Install a sample tube on the SIP to ensure that the outersleeveintentionallyhas been properlyThis pageleftinstalled.
blank
If the sleeve hits the bottom of the tube, loosen theretainer slightly and push the sleeve up as far as it willgo. Tighten the retainer.

Chapter 4: Maintenance

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5
Optimizing cytometer settings
This chapter covers the following topics:•

Cytometer settings workflow (page 64)•

Verifying the configuration and user preferences (page 66)•

Running a performance check (page 68)•

Setting up an experiment (page 72)•

Creating application settings (page 76)•

Recording compensation controls (page 79)•

Calculating compensation (page 82)

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Cytometer settings workflow
Introduction

This topic describes how to optimize cytometer settings. The
optimization is performed using the Cytometer Setup andTracking, Application Settings, and Compensation Setup featuresof BD FACSDiva software.

When to optimize
settings

Before you record data for a sample, optimize the cytometersettings for the sample type and fluorochromes used.

Manualcompensation

Compensation setup automatically calculates compensation
settings. If you choose to perform compensation manually, not allof the following instructions apply. For detailed instructions, see
the BD FACSDiva Software Reference Manual.

First-time users

If you are performing the procedures in this workflow for the firsttime, you should be familiar with BD FACSDiva softwareconcepts: workspace components, cytometer and acquisition
controls, and tools for data analysis.
For additional details, see the BD FACSDiva Software Reference
Manual.

Before you begin

Start the special order BD LSRFortessa X-20 cell analyzer andperform the setup and QC procedures. See Cytometer setup(page 29).

Workflow for
Cytometer optimization consists of the following steps.
optimizing settings
Step

Description

1

Verifying the configuration and user preferences (page 66)

2

Running a performance check (page 68)

3

Setting up an experiment (page 72)

Chapter 5: Optimizing cytometer settings

Step

Description

4

Creating application settings (page 76)

5

Recording compensation controls (page 79)6

Calculating compensation (page 82)

Note: Application settings are optional and do not have to be
saved for the experiments. However, they are useful for optimizing
cytometer settings.
About theexamplesThe examples in this chapter use a 4-color bead sample with thefollowing fluorochromes:
•

FITC

•

PE

•

PerCP-Cy™5.5•

APC

If you follow this workflow with a different bead sample (oranother sample type), your software views, data plots, andstatistics might differ from the example. Additionally, you mightneed to modify some of the instructions in the procedure.
The information shown in italics is for example only. You can
substitute your own names for folders and experiments.

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Verifying the configuration and user preferences
Introduction

This topic describes how to verify the cytometer configuration anduser preferences before you create an experiment.
Caution! To obtain accurate data results, the current
cytometer configuration must reflect yourBD LSRFortessa X-20 cell analyzer optics.

Procedure

To verify the configuration and preferences before you create anexperiment:
1. Select Cytometer > View Configurations and verify the current
configuration.

Your cytometer might include only the base configuration
when your cytometer is installed. You can create additional
configurations later as needed.
In this example, the cytometer configuration must include thefollowing parameters: FITC, PE, PerCP-Cy5.5, and APC.

Chapter 5: Optimizing cytometer settings

2. If you need to select a configuration other than the current
configuration:a. In the Configurations tab, select a configuration.
b. Click Set Configuration.
c. Click OK.
d. Verify that the configuration you just set matches yourBD LSRFortessa X-20 cell analyzer optics.
3. Click OK to close the Cytometer Configuration window.
4. Select File > Exit to close CS&T.
5. Select Edit > User Preferences.
6. Click the General tab and select the Load data after recording
checkbox.
See the BD FACSDiva Software Reference Manual for more
information about cytometer configurations and user preferences.
Next step

Running a performance check (page 68)

More information•

Setting up an experiment (page 72)

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Running a performance check
Introduction

This topic describes how to run a performance check as part ofquality control.

Overview

The CS&T application is designed to monitor performance on adaily basis and to optimize laser delay.
Running a performance check on a regular basis provides a
standard for monitoring changes in performance due todegradation of laser power, aging of PMTs, and other potential
cytometer service issues. Performance results are also affected by
fluidics performance. We strongly recommend following thefluidics maintenance procedures as described in Cleaning thefluidics (page 47).

Considerations

Some BP filters might not be normalized to CS&T settings. In thiscase, CS&T will generate Qr and Br numbers that are notcomparable from instrument to instrument. They are however, still
trackable on one cytometer. Part of the process for optimizing
cytometer settings includes verifying PMT voltages set by CS&T
for all parameters. Carefully examine any channel with a nonCS&T normalized filter.

Before you begin

Define the performance baseline for any configuration before
running a performance check. See Custom configurations andbaselines (page 43).

Chapter 5: Optimizing cytometer settings

ProcedureTo run a performance check:
1. Select Cytometer > CST.
2. Verify that the bead lot information under Setup Beads
matches the Cytometer Setup and Tracking bead lot.

3. Verify that the cytometer configuration is correct for yourexperiment.

If the cytometer is not set to the correct configuration:
a. Click Select Configuration in the Setup Control window.
b. Select the correct configuration from the list.
c. Click Set Configuration and then click OK.
4. Verify that the current configuration has a valid baseline
defined.
If not, see the BD Cytometer Setup and Tracking Application
Guide for more information on defining a baseline.
5. Prepare the CS&T beads according to the technical data sheet
provided with the beads or available on the BD Biosciences
website (bdbiosciences.com).

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6. Install the bead tube onto the SIP.
7. In the Setup Control window, select Check Performance fromthe Characterize menu.

8. Click Run.
9. Ensure that the SAMPLE FINE ADJ knob is set to the
midpoint and press RUN and LOW.
Plots appear under the Setup tab and the performance check isrun. The performance check takes approximately 5 minutes tocomplete.
10. Once the performance check is complete, click View Report.
11. Verify that the cytometer performance passed.
In the Setup tab, the cytometer performance results shouldhave a green checkbox displayed and the word Passed next toit.

If any parameters did not pass, see the BD Cytometer Setup
and Tracking Application Guide for troubleshooting
information.

Chapter 5: Optimizing cytometer settings

12. Select File > Exit to close the CS&T window and return to the
BD FACSDiva interface.
The CST Mismatch dialog opens.

Click the Details button to verify which cytometer settings will
be updated.
13. Click Use CST Settings.
By selecting Use CST Settings, the laser delay, area scaling, andother cytometer settings will be updated to the latest settingsfrom the performance check.
Next step

Continue the optimization of your cytometer for an experiment or
sample type as described in Setting up an experiment (page 72).

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Setting up an experimentIntroduction

This topic describes how to create an experiment in a new folder,specify the parameters of the experiment, and add compensation
tubes.

Creating anexperiment

To create an experiment:
1. Click the buttons on the Workspace toolbar to display thefollowing windows as needed:• Browser• Cytometer• Inspector• Worksheet• Acquisition Dashboard
When you add elements or make selections in the Browser, theInspector displays details, properties, and options that
correspond to your selection.
2. Click the New Folder button on the Browser toolbar to add a
new folder.
3. Click the folder and rename it MyFolder.
4. Click MyFolder, then click the New Experiment button on the
Browser toolbar.

a. Click the new experiment in the Browser and rename it
MyExperiment.

Chapter 5: Optimizing cytometer settings

5. Select MyExperiment in the Browser.
The Inspector displays details for the experiment.
Specifying
parameters

To specify the parameters for the new experiment:
1. Select Cytometer Settings for the experiment in the Browser.

Cytometer settings appear in the Inspector.

2. Make sure the parameters you need appear on the Parameters
tab in the Inspector.
If more than one parameter is available for a particular PMT,
you might have to select the one you need from a menu. For
example, you can set Detector F for the blue laser as FITC or
Alexa Fluor™ 488.

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a. Click the Parameter name to display the available
fluorochromes in the Parameters list.

b. Select the specific parameter from the menu. Your selection
appears as the selected parameter.

c. For this example, select FITC from the menu.
3. Delete any unnecessary parameters.

Chapter 5: Optimizing cytometer settings

a. Click the selection button (to the left of the parametername) to select the parameter.

b. Click Delete.
The parameter is deleted.

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Creating application settings
Introduction

This topic describes how to create application settings.

About application
settings

Application settings are associated with a cytometer configurationand include the parameters for the application, area scaling values,
PMT voltages, and threshold values, but not compensation. Eachtime a performance check is run for a configuration, theapplication settings associated with that configuration are updated
to the latest run.
Using application settings provides a consistent and reproducible
way to reuse cytometer settings for commonly used applications.
You can include area scaling adjustment in your application
settings. See Adjusting area scaling (page 169) for more
information.

Before you begin

Perform the cytometer setup procedure and run a performancecheck for the configuration that will be used for the application.

Procedure

To create application settings:
1. In the open experiment, right-click Cytometer Settings in the
Browser, then select Application Settings > Create Worksheet.
A second global worksheet is added with the plots createdaccording to the selections in the Parameters tab.

Chapter 5: Optimizing cytometer settings

Use the gray boxes and crosshairs to guide your optimization.
2. Load the unstained control tube onto the cytometer.
3. In the Cytometer window, optimize the PMT voltages for theapplication.
• Optimize the FSC and SSC voltages to place the populationof interest on scale.
• Optimize the FSC threshold value to eliminate debris
without interfering with the population of interest.
• If needed, increase the fluorescence PMT voltages to place
the negative population within the gray boxes. Align thecenter of the negative population with the crosshair visible
in the gray box.
Note: Do not decrease the fluorescence PMT voltages. Doingso can make it difficult to resolve dim populations from thenegative population.
4. Unload the unstained control tube from the cytometer.
5. Load the multicolor sample onto the cytometer or load singlecolor control tubes and verify each fluorochrome signal
separately.

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6. Verify that the positive populations are on scale.
If a positive population is off scale, lower the PMT voltage forthat parameter until the positive population can be seen
entirely on scale.
7. Unload the multicolor sample.
8. Place a tube containing DI water on the SIP and put thecytometer on standby.
9. (Optional) Save the application settings by right-clicking
Cytometer settings in the Browser, then selecting Application
Settings > Save.
10. In the Save Application Settings dialog, enter a descriptive
name for the application settings.

11. Click OK.
The application settings are saved to the catalog.
Next step

Recording compensation controls (page 79)

Chapter 5: Optimizing cytometer settings

Recording compensation controls
Introduction

This topic describes how to create and record compensation
controls using the Compensation Setup feature of BD FACSDiva
software and an experiment with optimized settings.

Creating
compensation
tubes

To create compensation control tubes:
1. Select Experiment > Compensation Setup >Create Compensation Controls.
The Create Compensation Controls dialog opens.
For this bead example, you do not need to provide non-generictube labels.

2. Click OK.

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Compensation control tubes are added to the experiment.
Worksheets containing appropriate plots and gates are added
for each compensation tube.

Recordingcompensation
settings

To record compensation settings:
1. Press RUN and HIGH on the cytometer fluid control panel.
2. Install the unstained control tube onto the SIP.
3. Expand the Compensation Controls specimen in the Browser.
4. Set the current tube pointer to the unstained control tube (itbecomes green), then click Acquire Data in the Acquisition
Dashboard.
5. Verify that the population of interest is displayed appropriately
on the FSC vs SSC plot and adjust voltages if necessary.

Chapter 5: Optimizing cytometer settings

Since the application settings have been optimized for yoursample, the cytometer settings should not require adjustment
other than the changing of FSC and SSC voltages to place thebeads on scale.
6. Adjust the P1 gate to surround only the singlets.
7. Right-click the P1 gate and select Apply to All Compensation
Controls.
The P1 gate on each stained control worksheet is updated withyour changes.
8. Click Record Data.
9. When recording is finished, remove the unstained control tube
from the cytometer.
10. Click Next Tube.
Caution! Do not change the PMT voltages after the
first compensation control has been recorded. Inorder to calculate compensation, all controls must
be recorded with the same PMT voltage settings. If
you need to adjust the PMT voltage for asubsequent compensation control, you must record
all compensation controls again.
11. Install the next tube onto the cytometer and repeat steps 8through 10 until data for all stained control tubes has beenrecorded.
12. Double-click the first stained control tube to display thecorresponding worksheet.

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13. Verify that the snap-to interval gate encompasses the positive
population.

14. Repeat steps 12 and 13 for the remaining compensation tubes.
Next step

After you have recorded data for each single-stained control,
calculate compensation as described in Calculating compensation(page 82).

Calculating compensation
Introduction

This topic describes how to calculate compensation.

Before you begin

Before you can calculate compensation, you need to record thedata for each single-stained control.

Procedure

To calculate compensation:
1. Select Experiment > Compensation Setup > Calculate
Compensation.
Note: If the calculation is successful, a dialog prompts you toenter a name for the compensation setup. The default name isyear/month/day/time.

Chapter 5: Optimizing cytometer settings

2. Enter a setup name and click Link & Save.
The compensation is linked to the cytometer settings and saved
to the catalog.
To help track compensation setups, include the experimentname, date, or both in the setup name.
The compensation setup is linked to the MyExperimentcytometer settings, and subsequent acquisitions in
MyExperiment are performed with the new compensation
settings.
We recommend that you always visually and statistically inspect
automatically calculated spectral overlap values. The means of thepositive controls should be aligned with the means of the negative
controls.
More information•

Recording compensation controls (page 79)83This page intentionally left blank6
Recording and analyzing data
This chapter covers the following topics:•

Data recording and analysis workflow (page 86)•

Preparing the workspace (page 87)•

Recording data (page 88)•

Analyzing data (page 91)•

Reusing an analysis (page 97)

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Data recording and analysis workflow
Introduction

This topic outlines the basic acquisition and analysis tasks using
BD FACSDiva software.

About theexamplesThe examples in this chapter are from two 4-color bead samples
with the following fluorochromes:
•

FITC

•

PE

•

PerCP-Cy5.5•

APC

If you use a different sample type or if you have skipped the
optimization steps in Optimizing cytometer settings (page 63),
your software window content, names of folders and experiments,
and your data plots and statistics might differ from those shown
here. You might also need to modify some of the instructions in theprocedure.
For additional details on completing some of the following steps,
see the BD FACSDiva Software Reference Manual.
This procedure builds on the results obtained in Optimizing
cytometer settings (page 63).
Workflow forrecording andanalyzing data

Recording and analyzing data consists of the following steps.
Step

Description

1

Preparing the workspace (page 87)

2

Recording data (page 88)

3Analyzing data (page 91)

4

Reusing an analysis (page 97)

Chapter 6: Recording and analyzing data

Preparing the workspace
Introduction

This topic describes how to prepare the workspace and apply
application settings to your experiment before recording data.

Procedure

To prepare the workspace:
1. Using the Browser toolbar, create a new specimen in
MyExperiment and rename it FourColorBeads.
2. Create two tubes for the FourColorBeads specimen. Rename
the tubes Beads_001 and Beads_002.
3. Expand the Global Worksheets folder in MyExperiment toaccess the default global worksheet, and rename the worksheet
MyData.
4. On the MyData worksheet, create the following plots forpreviewing the data:• FSC vs SSC• FITC vs PE• FITC vs PerCP-Cy5.5• FITC vs APC

Applying saved
application settings
to a newexperiment

When applications settings are applied to an experiment, thecytometer settings are updated with the parameters included in theapplication settings, optimized PMT voltages, threshold settings,
area scaling factors, and window extension values.
To apply saved application settings to your experiment:
1. Right-click the experiment-level Cytometer Settings and selectApplication Settings > Apply.

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2. In the Application Settings catalog, select the application
settings file you saved previously and click Apply.
If the parameters are not the same, a mismatch dialog opens.
• Click Overwrite to update all settings.
• Click Apply to change only the common parameters.
For more information, see the BD FACSDiva Software
Reference Manual.
The cytometer settings are renamed application settings and thecytometer settings icon in the Browser changes.
More information•

Creating application settings (page 76)•

Recording data (page 88)

Recording data
Introduction

This topic provides an example of how to preview and record data
for multiple samples.

Before you begin

Prepare the sample tubes.

Recording data

To record data:
1. Press RUN and HIGH on the cytometer fluid control panel.
2. Install the first sample tube onto the SIP.
3. Set the current tube pointer to Beads_001.
4. Click Acquire Data in the Acquisition Dashboard to begin
acquisition.

Chapter 6: Recording and analyzing data

5. While data is being acquired:a. Draw a gate around the singlets on the FSC vs SSC plot.
b. Rename the P1 gate to Singlets.
c. Use the Inspector to set the other plots to show only thesinglet population by selecting the Singlets checkbox.

6. Click Record Data.
7. When event recording has completed, remove the first tube
from the cytometer.
The MyData worksheet plots should look like the following.

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8. Install the second sample tube onto the SIP.
9. Set the current tube pointer to Beads_002.
10. Click Acquire Data to begin acquisition.
11. Before recording, preview the data on the MyData worksheet
to verify that all expected populations are visible and the data
is similar to the previous sample.
12. Click Record Data.
13. When event recording has completed, remove the second tube
from the cytometer.
14. If you are recording more than two tubes, repeat steps 8through 13 for the remaining tubes.
15. Print the experiment-level cytometer settings by right-clicking
the Cytometer Settings icon in the Browser and selecting Print.
16. Install a tube of DI water onto the SIP.
17. Place the cytometer in standby mode.
More information•

Analyzing data (page 91)

Chapter 6: Recording and analyzing data

Analyzing data
Introduction

This topic describes how to analyze recorded tubes by creatingplots, gates, a population hierarchy, and statistics views on a new
global worksheet.

Analyzing data

To analyze data:
1. Use the Browser toolbar to create a new global worksheet.
Rename it MyDataAnalysis.
2. Create the following plots on the MyDataAnalysis worksheet:• FSC vs SSC• FITC vs PE• FITC vs PerCP-Cy5.5• FITC vs APC
3. Create a population hierarchy and a statistics view, and set
them below the plots on the worksheet.
• Right-click any plot and select Show Population Hierarchy.
• Right-click any plot and select Create Statistics View.
4. Set the current tube pointer to Beads_001.
5. Draw a gate around the singlets on the FSC vs SSC plot.
6. Use the population hierarchy to rename the population
Singlets.

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7. Select all plots except the FSC vs SSC plot, and use the Plot tabin the Inspector to specify to show only the singlet population.

8. Select all plots, and click the Title tab in the Inspector.

Chapter 6: Recording and analyzing data

9. Select the Tube and Populations checkboxes to display theirnames in plot titles.

10. On all fluorescence plots:• Make all plots biexponential. Select all fluorescence plots
and select the X Axis and Y Axis checkboxes in the Plot tabof the Inspector.

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BD LSRFortessa X-20 Cell Analyzer User’s Guide• In the FITC vs PE plot, draw a gate around the
FITC-positive population. Name the population FITC
positive in the population hierarchy.
• In the FITC vs PE plot, draw a gate around the PE-positive
population. Name the population PE positive in the
population hierarchy.
• In the FITC vs PerCP-Cy5.5 plot, draw a gate around the
PerCP-Cy5.5-positive population. Name the population
PerCP-Cy5.5 positive in the population hierarchy.
• In the FITC vs APC plot, draw a gate around the APCpositive population. Name the population APC positive inthe population hierarchy.
11. Format the statistics view.
a. Right-click the statistics view and select Edit Statistics
View.
b. Click the Header tab and select the Specimen Name andTube Name checkboxes.
c. Click the Populations tab and select all populations except
All Events. Clear the %Parent, %Total, and #Events
checkboxes.
d. Click the Statistics tab and select the mean for each of thefluorescence parameters.

Chapter 6: Recording and analyzing datae. Click OK.
12. Print the analysis.

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Your global worksheet analysis objects should look like thefollowing.

Chapter 6: Recording and analyzing data

More information•

Reusing an analysis (page 97)

Reusing an analysis
Introduction

This topic describes how to use a global worksheets to apply thesame analysis to a series of recorded tubes. Once you define ananalysis for a tube, you can use it to analyze the remaining tubes inthe experiment. After viewing the data, print the analysis or save it
to a normal worksheet.

Reusing an analysis To reuse the analysis:
1. Set the current tube pointer to the Beads_002 tube.
2. View the Beads_002 data on your analysis worksheet. Adjust
the gates as needed.
Adjustments apply to subsequent tubes viewed on theworksheet. To avoid altering a global worksheet, save ananalysis to a normal worksheet, then make adjustments on thenormal worksheet.
3. Print the analysis.
Saving the analysis

When you perform analysis with a global worksheet, the analysis
does not save with the tube.
If you define your analysis on a global worksheet before recording
data, you can specify to automatically save the analysis afterrecording data. You set this option in User Preferences.
To save a copy of the analysis with a tube:
1. Expand the MyDataAnalysis global worksheet icon in the
Browser.

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2. Right-click its analysis and select Copy.

3. Click the Worksheets View button on the Worksheet toolbar toswitch to the normal worksheet view.
4. Select Worksheet > New Worksheet to create a new normal
worksheet.
5. Right-click the Beads_001 tube icon in the Browser, and selectPaste.

The analysis objects from the MyDataAnalysis global
worksheet are copied to the Beads_001_Analysis normal
worksheet. Double-click the Beads_001 tube in the Browser toview the analysis.
Applying ananalysis to normal
worksheets

You can apply the global worksheet analysis to multiple tubes (ona single normal worksheet) by selecting multiple tubes before
pasting the analysis. Ensure that you collapse all tube elements in
the Browser before you paste them to multiple tubes.

More information•

Analyzing data (page 91)

7Technical overview
This chapter provides a technical overview of the following topics:•

About fluidics (page 100)•

About optics (page 101)•

About electronics (page 112)

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About fluidics
Introduction

This topic describes the fluidics system in the special order
BD LSRFortessa X-20 cell analyzer.

Pressure-driven
fluidics system

The fluidics system in the BD LSRFortessa X-20 cell analyzer ispressure-driven. A built-in air pump provides a sheath pressure of
5.5 psi. After passing through the sheath filter, sheath fluid is
introduced into the lower chamber of the quartz flow cell.

Hydrodynamic
focusing

The sample to be analyzed arrives in a separate pressurized stream.
When a sample tube is placed on the SIP, the sample is forced up
and injected into the lower chamber of the flow cell by a slightoverpressure relative to the sheath fluid. The conical shape of thelower chamber creates a laminar sheath flow that carries the
sample core upward through the center of the flow cell, where theparticles to be measured are intercepted by the laser beam. This
process is known as hydrodynamic focusing.

Low sample
pressure(12 µL/min)

Sheathfluid

Laser beamSampleSheathfluid

High sample
pressure(60 µL/min)

Sheathfluid

Laser beamSampleSheathfluid

The objective in flow cytometric analysis is to have at most one cellor particle moving through a laser beam at a given time. The
difference in pressure between the sample stream and sheath fluid
stream can be used to vary the diameter of the sample core.

Chapter 7: Technical overview

Increasing the sample pressure increases the sample flow rate
thereby increasing the core diameter. The flow rate should be set
according to the type of application you are running.
•

A higher flow rate is generally used for qualitative
measurements such as immunophenotyping. The data is lessresolved, but is acquired more quickly.

•

A lower flow rate is generally used in applications where
greater resolution and quantitative measurements are critical,
such as DNA analysis.

Proper operation of fluidic components is critical for particles tointercept the laser beam properly. Always ensure that the fluidics
system is free of air bubbles and debris, and is properly
pressurized.

About optics
Introduction

Optics system

This topic describes the optics system and provides information
about:•

Light scatter (page 102)•

Fluorescence (page 102)•

Optical filter theory (page 104)•

Compensation theory (page 109)

The optics system consists of lasers, optical filters, and detectors.
Lasers illuminate the cells or particles in the sample and opticalfilters direct the resulting light scatter and fluorescence signals tothe appropriate detectors.

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Light scatter

When a cell or particle passes through a focused laser beam, laser
light is scattered in all directions. Light that scatters axial to thelaser beam is called forward scatter (FSC) and light that scattersperpendicular to the laser beam is called side scatter (SSC).
Side scatter (SSC)

Light source

Forward scatter (FSC)

FSC and SSC are related to certain physical properties of cells.

Fluorescence•

FSC. Indicates relative differences in the size of the cells or
particles. Larger cells scatter more light and therefore they arehigher in FSC.

•

SSC. Indicates relative differences in the internal complexity or
granularity of the cells or particles. More granular cells deflect
more light than less granular cells, and therefore are higher in
SSC.

When cells or particles stained with fluorochrome-conjugatedantibodies or other dyes pass through a laser beam, the dyes canabsorb photons (energy) and be promoted to an excited electronic
state. In returning to their ground state, the dyes release energy,
most of which is emitted as light. This light emission is known as
fluorescence.
Fluorescence is always a longer wavelength (lower-energy photon)than the excitation wavelength. The difference between the
excitation wavelength and the emission wavelength is known as
the Stokes shift. Some fluorescent compounds such as PerCP
exhibit a large Stokes shift, absorbing blue light (488 nm) andemitting red light (675 nm), while other fluorochromes such as

Chapter 7: Technical overview

FITC have a smaller Stokes shift, absorbing blue light (488 nm)
and emitting green light (530 nm).
The following figure shows the emission spectra of somecommonly used fluorochromes.

Actual emission intensity will depend on excitation wavelength.
See Fluorescence spectra (page 130) for more information on
excitation and emission of fluorochromes. An interactive spectral
viewer is also available at bdbiosciences.com.

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Optical filter theory Optical filters modify the spectral distribution of light scatter andfluorescence directed to the detectors. When photons encounter an
optical filter, they are either transmitted, absorbed, or reflected.

Photonstransmitted

Photonsabsorbed

Photonsreflected

Even though an optical filter is rated at its 50% transmission point,
the filter passes (lets through) a minimal amount of light outside ofthis indicated rating.
The slope of an optical filter transmission curve indicates filter
performance. A relatively steep slope indicates a high-performance,high-quality optical filter that provides deep attenuation of out-ofband wavelengths. A less steep slope indicates that more lightoutside the rated bandwidth is being transmitted.
Types of opticalfilters

There are four types of filters.
•

Longpass (LP) filters. Transmit wavelengths that are longer
than the specified value.

•

Shortpass (SP) filters. Transmit wavelengths that are shorter
than the specified value. This type of filter is notrecommended, but can be used in some custom configurations.
See Shortpass (SP) filters (page 106).

•

Bandpass (BP) filters. Pass a narrow spectral band of light by
combining the characteristics of shortpass filters, longpass
filters, and absorbing layers.

Chapter 7: Technical overview•

Notch filters. Pass all frequencies except those in a stop band
centered on a center frequency. They are the opposite of
bandpass filters.

The BD LSRFortessa X-20 uses LP filters and BP filters. Notch
filters are sometimes used in special order BD LSRFortessa X-20
instruments.
LP filters pass wavelengths longer than the filter rating. For
example, a 500-LP filter permits wavelengths 500 nm or longer topass through it and either absorbs or reflects wavelengths shorter
than 500 nm.
Longpass% Transmission

Longpass (LP)filters

Wavelength (nm)105BD LSRFortessa X-20 Cell Analyzer User’s Guide

Shortpass (SP)filters

An SP filter has the opposite properties of an LP filter. An SP filter
passes light with a shorter wavelength than the filter rating. For
example, a 500-SP filter passes wavelengths of 500 nm or shorter,and reflects or absorbs wavelengths longer than 500 nm.
Shortpass% Transmission

106

Wavelength (nm)

Chapter 7: Technical overview

Bandpass (BP)filters

A BP filter transmits a relatively narrow range or band of light. BP
filters are typically designated by two numbers. The first number
indicates the center wavelength and the second refers to the widthof the band of light that is passed. For example, a 500/50-BP filtertransmits light that is centered at 500 nm and has a total
bandwidth of 50 nm. Therefore, this filter transmits light between
475 and 525 nm.

% Transmission

Bandpass

Wavelength (nm)The performance of an optical BP filter depends on the opticaltransmission. Sample transmission curves are shown in thefollowing figure. A filter with a narrower (steeper) transmission
curve generally yields higher performance. The transmission
specifications depend on the construction of the filter.
Higher performance filters generally have multiple layers of opticalcoatings and unique manufacturing processes. For moredemanding multicolor applications, higher performance filters areavailable through the special order research program. Contact
your local BD Biosciences sales representative for details.

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BD LSRFortessa X-20 Cell Analyzer User’s Guide% Transmission

108

Wavelength (nm)Dichroic mirrors

Dichroic filters that are used to direct different color light signals
to different detectors are called dichroic mirrors.
Although some of the properties of LP and SP filters are similar todichroic mirrors (for example, allowing a specific wavelength
range to pass), filters and mirrors cannot be used interchangeably,
especially if used as dichroic mirrors. A dichroic mirror must havea surface coating that reflects certain wavelengths, but many LP or
SP filters are absorbance filters that do not have any specificreflective characteristics. Also, optical filters and dichroic mirrors
are rated at a specific angle of incidence. When used in front of thefluorescence detectors, they are perpendicular to the incident light,
and when used as a dichroic mirror, they are placed at an angle
relative to the light source. Their optical properties are therefore
designed for that angle of incidence.

Chapter 7: Technical overview

Fluorochromes emit light over a range of wavelengths. Opticalfilters are used to limit the range of frequencies measured by agiven detector. However, when two or more fluorochromes areused, the overlap in wavelength ranges often makes it impossible
for optical filters to isolate light from a given fluorochrome. As aresult, light emitted from one fluorochrome appears in a detector
intended for another. This is referred to as spillover. Spillover canbe corrected mathematically by using a method called
compensation.
In the following example, FITC emission appears primarily in the
FITC detector, but some of its fluorescence spills over into the PE
detector. The spillover must be corrected or compensated for.
Alternatively, the spillover can be minimized by discrete excitationof fluorochromes. In the following example, excitation with a 561nm laser (special order) will help minimize spillover.

Normalized intensity

Compensation
theoryWavelength (nm)This spillover can be seen in a dot plot of FITC vs PE. The FITC
spillover in the PE detector must be corrected as demonstrated in
the two figures that follow.

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FITC-positive
population

PE

Unstained
particles
FITC

Using the Compensation tab of the Cytometer window in
BD FACSDiva software, you can adjust the PE-%FITC spectral
overlap value. Compensation is optimal when the positive andnegative FITC populations have the same means in the PE
parameter statistics.
The following image shows the FITC spillover optimallycompensated out of the PE parameter.

FITC-positive
population

PE
Unstained
particles

Matching means
FITC

Once fluorescence compensation has been set for any sample, thecompensation setting remains valid for a subsequent dim or brightsample (provided the signal is not saturated), because
compensation subtracts a percentage of the fluorescence intensity.
The following figure illustrates this principle. Although the signals
differ in intensity, the percentage of the FITC spillover into the PE
detector remains constant.

Chapter 7: Technical overview

FITC

PE

Normalized intensity

Different intensity FITC signals

Same proportion or percentage ofspectral overlap in PE channel

Wavelength (nm)111

BD LSRFortessa X-20 Cell Analyzer User’s Guide

About electronics
Introduction

This topic describes the electronics in the special order
BD LSRFortessa X-20 cell analyzer.

Pulse

As cells or other particles pass through a focused laser beam, theyscatter the laser light and can emit fluorescence. Because the laser
beam is focused on a small spot and particles move rapidly throughthe flow cell, the scatter or fluorescence emission signal has a very
brief duration—only a few microseconds. This brief flash of light isconverted into an electrical signal by the detectors. The electricalsignal is called a pulse. The following figures illustrate the anatomy
of a pulse.

Signal intensity

1. A pulse begins when a particle enters the laser beam. At thispoint, both the beam intensity and signal intensity are low.

Time

2. The pulse reaches a maximum intensity or height when theparticle reaches the middle of the beam, where the beam andsignal intensity are the brightest. The peak intensity, or heightof the pulse, is measured at this point.

Signal intensity

112

Time

Chapter 7: Technical overview

Signal intensity

3. As the particle leaves the beam, the pulse trails off below thethreshold.

Time

Pulse
measurements

The pulse processors measure pulses by three characteristics:
height, area, and width.

•

Height. The maximum digitized intensity measured for thepulse.

•

Area. The integration of all the digitized samples over time,
where time is the window gate plus 1/2 the window extension
added before the initial threshold, plus the other half of thewindow extension value added after the pulse drops below thethreshold. The window gate extends until the pulse is 75% of
the initial threshold.

•

Width. Calculates: Area x 64,000
Height113

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Digital electronics

BD LSRFortessa X-20 electronics digitize the signal intensity
produced by a detector. The digitized data is stored in memory andfurther processed by the electronics to calculate:•

Pulse height, area, and width•

Compensation•

Parameter ratios

These results are transferred to your workstation computer for
further processing by BD FACSDiva software. For more
information about digital theory, see Digital Theory in the
BD FACSDiva Software Reference Manual.
Threshold

The threshold is the level at which the system starts to measuresignal pulses. A threshold is defined for a specific detector signal.
The system continuously samples the digitized signal data andcalculates pulse area, height, and width for all channels based on
the time interval during which the threshold is exceeded.
Thresholds can also be set for more than one parameter, and pulse
measures are based on either of the following:•

Intervals during which ALL signals exceed their thresholdvalue (AND threshold in the software)•Intervals during which ANY signal exceeds its threshold value(OR threshold in the software)Chapter 7: Technical overview

Laser controls

Controls in the Laser tab of the Cytometer window are used tomanually set the (laser) delay, area scaling, and window extension
values.
These parameters are set by BD Biosciences service personnel
during instrument installation and performance check and are
updated each time you run a performance check.
If needed, see Optimizing laser delay (page 167) for instructions on
manually adjusting laser delay settings. Do not otherwise change
the settings in the Laser tab unless instructed to do so by
BD Biosciences. Changing the settings affects your data.

More information•

Running a performance check (page 68)•

Optimizing laser delay (page 167)

115

This page intentionally left blank8
TroubleshootingThis chapter covers the following topics:•

Cytometer troubleshooting (page 118)•

Electronics troubleshooting (page 127)

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Cytometer troubleshooting
Introduction

Droplets are visibleon the SIPThis topic describes possible problems and recommended solutions
for special order BD LSRFortessa X-20 cell analyzer issues.

Possible causes

Recommended solutions

Worn O-ring in the retainer

Replace the O-ring. See Changing thesample tube O-ring (page 60).

Outer sleeve is not seated in
the retainer

1. Loosen the retainer.
2. Push the outer sleeve up into theretainer until seated.
3. Tighten the retainer.

Outer sleeve is not on thesample injection tube

Replace the outer sleeve.
1. Loosen the retainer.
2. Slide the outer sleeve over the
sample injection tube until it isseated.
3. Tighten the retainer.

Waste line is pinched,preventing proper aspiration

Check the waste line.

Waste tank is full

Empty the waste tank.

Droplet containment vacuum
is not functioning

Call your BD service representative.

The HTS acquisition control
switch is in plate mode

Change the acquisition control switch
to tube mode. See the BD HighThroughput Sampler User’s Guide formore information.

Chapter 8: Troubleshooting

Sample tube notfitting on SIP

Rapid sample
aspiration

No events inacquisition display
and RUN button is
green

Possible causes

Recommended solutions

Sample tube other than
Falcon® tubes used

Use Falcon 12 x 75-mm sample tubes.
See Equipment (page 178).

Worn Bal seal

Replace the Bal seal. See Changing the
Bal seal (page 58).

Sample tube is cracked

Transfer contents to a new tube.

Possible causes

Recommended solutions

Support arm is to the side

Place the support arm under the
sample tube.

Droplet containment module
is failing

Try the solutions in Droplets arevisible on the SIP (page 118). If theissue is not resolved, call your BD
service representative.

Possible causes

Recommended solutions

Threshold is not set to thecorrect parameter (usually
FSC)

Set the threshold to the correct
parameter for your application.

Threshold level is too highLower the threshold level.

PMT voltage for thresholdparameter is set too low

Set the PMT voltage higher for thethreshold parameter.

Gating issue

See the BD FACSDiva Software
Reference Manual for information on
setting gates.

Air in the sheath filter

Purge the filter. See Removing air
bubbles (page 33).

Air bubble or debris in theflow cell

Prime the fluidics system. See Priming
the fluidics (page 39).

119

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Possible causes

Recommended solutions

No sample in the tube

Verify that sample remains in the tube
and if necessary, add sample to thetube or install a new sample tube.

Sample is not mixed properly

Mix the sample to suspend the cells.

Waste tank is full

Empty the waste tank.

PMT voltages set too low or
too high for display
parameter

Adjust the PMT voltages.

Too few events are displayed

Increase the number of events todisplay.

Sample injection tube is
cloggedRemove the sample tube to allow
backflushing.
If the event rate is still erratic, clean
the sample injection tube. See Cleaning
the fluidics (page 47).

Bal seal is worn

Replace the Bal seal. See Changing the
Bal seal (page 58).

Instrument is not warmed up

Wait 30 minutes for the instrument towarm up.

Laser delay is set incorrectly

Adjust the laser delay settings. See
Manual settings (page 165).

Laser is not functioning

Verify the malfunction by changing thethreshold to an alternative laser whilerunning the appropriate sample. If
unsuccessful, contact BD Biosciences.

Tube is cracked or misshapen

Replace the sample tube.

Chapter 8: Troubleshooting

No events inacquisition display
and RUN button isorangePossible causes

Recommended solutions

RUN is not activatedPress the RUN button.

Sample tube is not installed
or is not properly seated

Install the sample tube correctly on the
SIP.

Waste container ispressurized

Replace the waste air filter.
Caution! Pressurized contents
might spray. Use appropriate
cautionary measures.
1. Slowly loosen the waste tank cap
to relieve the pressure in the waste
tank.
2. Remove the waste vent filter byrotating counter-clockwise.
3. Install a new waste vent filter.

Sample tube is cracked

Replace the sample tube.

Waste tubing line is notconnected to the waste cap

Connect the waste tubing line to thewaste cap.

Sheath container is notpressurizedEnsure that the sheath container lid
and all connectors are securely
seated.

Inspect the sheath container O-ring
inside the lid and replace it ifnecessary.

Bal seal is worn

Replace the Bal seal. See Changing the
Bal seal (page 58).

Air leak at sheath container

Ensure that the sheath container lid
and all connectors are securely seated.

Sheath container is empty

Fill the sheath container.

Air in sheath filter

Purge the filter. See Removing air
bubbles (page 33).

121

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No fluorescencesignalNo signal in red
laser channels

Possible causes

Recommended solutions

Incorrect fluorochrome
assignment

Make sure that the cytometer
configuration in the software matches
the optical filters in the cytometer andthe configuration is as expected.

Wrong filter is installed

Make sure the appropriate filter isinstalled for each fluorochrome. See
Changing optical filters and mirrors(page 42).

Laser is not functioning

Call your BD service representative.

Possible causes

Recommended solutions

Incorrect laser delays due toa change in the sheath tankfluid level

Check the fluid level in the sheathtank and refill if necessary.

Adjust the laser delay settings. See
Manual settings (page 165).

Possible causes

Recommended solutions

Air bubbles in the sheathfilter or flow cell

Remove the air bubbles. See Removing
air bubbles (page 33).

Threshold level is too low

Increase the threshold level. See theBD FACSDiva Software Reference
Manual for instructions.

PMT voltage for thethreshold parameter is set
too high

Set the PMT voltage lower for thethreshold parameter. See theBD FACSDiva Software Reference
Manual for instructions.

Sample is too concentrated

Dilute the sample.

Sample flow rate is set to
HIGH

Set the sample flow rate to MED or
LOW.

High event rate

Chapter 8: Troubleshooting

Low event rate
Possible causes

Recommended solutions

Threshold level is too highLower the threshold level. See theBD FACSDiva Software Reference
Manual for instructions.

Air bubble or debris in theflow cell

Prime the fluidics system. See Priming
the fluidics (page 39).

PMT voltage for thethreshold parameter is set
too low

Set the PMT voltage higher for thethreshold parameter. See theBD FACSDiva Software Reference
Manual for instructions.

Sample is not adequately
mixed

Mix the sample to suspend the cells.

Sample is too diluted

Concentrate the sample. If the flowrate setting is not critical to theapplication, set the flow rate switch to
MED or HI.

Sample injection tube is
cloggedRemove the sample tube to allow
backflushing.
If the event rate is still erratic, clean
the sample injection tube. See Cleaning
the fluidics (page 47).

Erratic event rate
Possible causes

Recommended solutions

Sample tube is cracked

Replace the sample tube.

Air bubble or debris in theflow cell

Prime the fluidics system. See Priming
the fluidics (page 39).

Bal seal is worn

Replace the Bal seal. See Changing the
Bal seal (page 58).

123

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Possible causes

Recommended solutions

Sample injection tube is
cloggedRemove the sample tube to allow
backflushing.
If the event rate is still erratic, clean
the sample injection tube. See Cleaning
the fluidics (page 47).

Distorted scatterparameters

Excessive amountof debris in display

Contaminated sample

Prepare the specimen again. Ensurethat the tube is clean.

Sheath filter is dirty

Replace the filter. See Changing thesheath filter (page 56).

Possible causes

Recommended solutions

Cytometer settings are
improperly adjusted

Optimize the scatter parameters. See
the BD FACSDiva Software Reference
Manual for instructions.

Air bubble in the sheath filter
or flow cell

Purge the air from the filter. See
Removing air bubbles (page 33).

Flow cell is dirty

Flush the system. See Flushing thesystem (page 50).

Air leak at sheath container

Ensure that the sheath container lid istight and all connectors are secure.

Hypertonic buffers or
fixative

Replace the buffers or fixative.

Possible causes

Recommended solutions

Threshold level is too low

Increase the threshold level.

Sheath filter is dirty

Replace the filter. See Changing thesheath filter (page 56).

Flow cell is dirty

Flush the system. See Flushing thesystem (page 50).

Chapter 8: TroubleshootingHigh CV or poor QC
results

Possible causes

Recommended solutions

Dead cells or debris in thesampleExamine the sample under amicroscope.

Sample is contaminatedRe-stain the sample. Ensure that the
tube is clean.

Stock sheath fluid iscontaminatedRinse the sheath container with DI
water, then fill the container withsheath fluid from another (or new lot)bulk container.

Possible causes

Recommended solutions

Air bubble in sheath filter orflow cell

Purge the filter. See Removing air
bubbles (page 33).

Prime the fluidics system. See
Priming the fluidics (page 39).

Sample flow rate is set too
high

Set the sample flow rate lower.

Air leak at sheath container

Ensure that the sheath container lid istight and all connectors are secure.

Flow cell is dirty

Flush the system. See Flushing thesystem (page 50).

The dichroic mirrors are
installed backwards

Remove the dichroic mirrors, reverse
them, and replace them in the opticalholder.

125

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Possible causes

Recommended solutions

Waste tank is pressurized

Replace the waste vent filter.
Caution! Pressurized contents
might spray. Use appropriate
cautionary measures.
1. Slowly loosen the waste tank cap
to relieve the pressure in the waste
tank.
2. Remove the waste vent filter byrotating counter-clockwise.
3. Install a new waste vent filter.

Poor sample preparation

Repeat sample preparation.

Sample was not diluted in thesame fluid as the sheath fluid

Dilute the sample in the same fluid as
you are using for sheath.

Optical filters are incorrect

Check the configuration and insert thecorrect filters.

Old or contaminated QC
particles

Make new QC samples and performthe quality control procedure again.

Instrument is not warmed up

Wait 30 minutes for the instrument towarm up.

Laser is not functioning

Contact BD Biosciences.

Optical alignment problem

Contact BD Biosciences.

Optical filters are incorrect

Check the configuration and insert thecorrect filters.

Chapter 8: Troubleshooting

Electronics troubleshooting
Introduction“Cytometer
Disconnected” incytometer window

This topic describes possible problems and recommended solutions
for BD LSRFortessa X-20 electronic issues.

Possible causes

Recommended solutions

Cytometer power is off

Turn on the cytometer main power.

Communication failure
between workstation andcytometer

1. In BD FACSDiva software, selectCytometer > Connect.
2. If connecting does not work,
restart the cytometer. Turn thecytometer off, wait 1 minute, andturn on the cytometer main power.
3. If connecting still does not work,
contact BD Biosciences.

127

This page intentionally left blank9
Detector array configurations
This chapter covers the following topics:•

Fluorescence spectra (page 130)•

About configuration maps (page 132)•

About the base configuration (page 133)•

Base configuration polygon maps (page 137)•

Special order configurations (page 150)

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Fluorescence spectra
Introduction

This topic shows sample emission spectra from common
fluorochromes, as well as the more common laser excitation lines.
This information is useful for designing multicolor panels. Aninteractive fluorescence viewer is also available atbdbiosciences.com.

Designing
multicolor panels

The special order BD LSRFortessa X-20 cell analyzer is designed
specifically for multicolor research. There are many options fordyes, reagents, and system configurations.
When choosing a configuration or panel, it is important toremember that final results depend on the excitation and emission
spectra of the individual dye, the number of fluorescently labeledbinding sites on the cell, as well as spectral overlap and spillover toother PMTs. For more information about designing multicolor
panels, see Selecting Reagents for Multicolor Flow Cytometry(Part No. 23-9538-02).

Chapter 9: Detector array configurations

Example laser anddye interactions

The following figure shows the emission spectra of some common
dyes, based on laser excitation. In many cases, a given dye can be
excited by multiple laser wavelengths, yielding different emission
intensities.
UV (355 nm)

Violet (405 nm)

Blue (488 nm)

Yellow-Green(561 nm)
Available only on
Special OrderResearch Products

Red (640 nm)

131

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

About configuration maps
Introduction

This topic describes the filter and mirror arrangements in thedetector arrays.

Filter and mirror
arrangement

The filters are arranged in the detector array to steer progressively
shorter wavelengths of light to the next PMT in the array. The
longest wavelength should be in the A position and the shortest
wavelength should be in the last position used.
There should not be any empty slots for any laser being used.
Always use a blank optic holder.
If a slot is filled with a filter or mirror, an identifying number
appears in that position on the configuration map. If a slot is filledwith a blank optic holder, that position on the configuration map isunlabeled.

Chapter 9: Detector array configurations

About the base configuration
Introduction

This section describes the base configuration options available
with the special order BD LSRFortessa X-20 cell analyzer.

Nine available
configurations

The base configuration for a BD LSRFortessa X-20 cell analyzer
supports detectors, filters, and mirrors for up to five lasers toprovide up to 18-color detection.

Numberof lasers

ColorsLasersSee section...

1

4

4 BlueFour-color blue laser configuration (page 139)

2

6

4 BlueFour-color blue laser configuration (page 139)

2 Red

Two-color red laser configuration (page 142)

5 BlueFive-color blue laser configuration (page 140)

2 NUV

Two-color NUV laser configuration (page 148)

5 BlueFive-color blue laser configuration (page 140)

3 Red

Three-color red laser configuration (page 143)

7

8

133

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Numberof lasers

ColorsLasersSee section...

3

8

4 BlueFour-color blue laser configuration (page 139)

2 Red

Two-color red laser configuration (page 142)

2 NUV

Two-color NUV laser configuration (page 148)

4 BlueFour-color blue laser configuration (page 139)

2 Red

Two-color red laser configuration (page 142)

2 VioletTwo-color violet laser configuration (page 145)

5 BlueFive-color blue laser configuration (page 140)

3 Red

Three-color red laser configuration (page 143)

2 NUV

Two-color NUV laser configuration (page 148)

5 BlueFive-color blue laser configuration (page 140)

3 Red

Three-color red laser configuration (page 143)

3 VioletThree-color violet laser configuration (page 146)

5 BlueFive-color blue laser configuration (page 140)

3 Red

Three-color red laser configuration (page 143)

6 VioletSix-color violet laser configuration (page 147)

8

10

11

14

Chapter 9: Detector array configurations

Numberof lasers

ColorsLasersSee section...

4

10

4 BlueFour-color blue laser configuration (page 139)

2 Red

Two-color red laser configuration (page 142)

2 VioletTwo-color violet laser configuration (page 145)

2 NUV

Two-color NUV laser configuration (page 148)

2 BlueTwo-color blue laser configuration (page 138)

3 Red

Three-color red laser configuration (page 143)

2 NUV

Two-color NUV laser configuration (page 148)

5 Yellow-green

Five-color yellow-green laser configuration (page 149)

5 BlueFive-color blue laser configuration (page 140)

3 Red

Three-color red laser configuration (page 143)

3 VioletThree-color violet laser configuration (page 146)

2 NUV

Two-color NUV laser configuration (page 148)

5 BlueFive-color blue laser configuration (page 140)

3 Red

Three-color red laser configuration (page 143)

6 VioletSix-color violet laser configuration (page 147)

2 NUV

Two-color NUV laser configuration (page 148)

6 BlueSix-color blue laser configuration (page 141)

4 Red

Four-color red laser configuration (page 144)

6 VioletSix-color violet laser configuration (page 147)

2 NUV

Two-color NUV laser configuration (page 148)

12

13

16

18

135

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Base configuration

The special order BD LSRFortessa X-20 cell analyzer has one baseconfiguration at installation. Custom configurations can be addedfor different applications. The following image shows a default
base cytometer configuration.

Chapter 9: Detector array configurations

Upgrade options

The special order BD LSRFortessa X-20 cell analyzer opticalconfiguration can be upgraded to use different special order
wavelength options and a variety of detectors. See Special orderconfigurations (page 150) for a list of some common special orderlaser/detector combinations available through the BD special orderresearch program. Many other special order configurations andoptions are also available.

More information•

Verifying the configuration and user preferences (page 66)•

Base configuration polygon maps (page 137)

Base configuration polygon maps
Introduction

This section describes how filters and mirrors are arranged forstandard polygon configurations.

About the maps

The tables in this section show the detectors, filters, and mirrors
used in each configuration, and recommended fluorochromes foreach detector. The word “blank” indicates that a blank opticalholder should be used instead of an optic holder containing amirror or filter. A dash (—) indicates that no slot exists for a mirror
in that PMT position.

137

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Two-color blue
laser configuration

The following map shows the two-color configuration for the
488-nm blue laser.
PMT

LP mirror

BP filter

FluorochromesA

640

695/40PerCP-Cy5.5

B505

525/50

FITC

C

Blank

488/10SSC

525/50
505LP

/40
488
695
/10
640LP

138

Chapter 9: Detector array configurations

The following map shows the four-color configuration for the
488-nm blue laser.
PMT

LP mirror

BP filter

Fluorochromes

A

750

780/60PE-Cy™7

B

685

695/40PerCP-Cy5.5, PE-Cy™5, PerCP, PIC

550

575/26PED

505

530/30

FITC, Alexa Fluor™ 488

E

Blank

488/10SSC

695/405350

6 8 5 LP

0/

5L

P

780

/60

575

/26

48

8/1

0

P

75 0 LP 5 5 0 L

Four-color blue
laser configuration

30

139

BD LSRFortessa X-20 Cell Analyzer User’s Guide

The following map shows the five-color configuration for the
488-nm blue laser.
PMT

LP mirror

BP filter

Fluorochromes

A

750

780/60PE-Cy7B

685

695/40PerCP-Cy5.5C

600

610/20

BD Horizon™ PE-CF594,
PE-Texas Red®

D

550

575/26PEE

505

530/30

FITC, Alexa Fluor™ 488

F

Blank

488/10SSC

695/4057

55685 LP

5/

26

0L

780

/60

0
0/3
53
LP
505

488/10

P

P

610

/20

Five-color blue
laser configuration

7 5 0 LP 600 L

140

Chapter 9: Detector array configurations

The following map shows the six-color configuration for the
488-nm blue laser.
PMT

LP mirror

BP filter

Fluorochromes

A

750

780/60PE-Cy7B

685

695/40PerCP-Cy5.5C

655

670/30PE-Cy5D

600

610/20PE-CF594, PE-Texas Red®

E

550

575/26PEF

505

530/30

FITC, Alexa Fluor™ 488

G

Blank

488/10SSC

695/400/20

0L
505 LP

530/30P
780/60

488/10
6
5/2
57
LP
55 0

61

60

685 LP

7 5 0 LP 655 L

P

670

/30

Six-color blue laser
configuration

141

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Two-color red laser The following map shows the two-color configuration for theconfiguration
640-nm red laser.
PMT

LP mirror

BP filter

Fluorochromes

A

750

780/60APC-Cy7, APC-H7

B

Blank

670/14APC

670/14/60780
7 5 0 LP

142

Chapter 9: Detector array configurations

The following map shows the three-color configuration for the
640-nm red laser.
PMT

LP mirror

BP filter

Fluorochromes

A

750

780/60APC-Cy7, APC-H7

B

710

730/45Alexa Fluor™ 700

C—670/14APC

730/45710 LP

/60
670
780
/14
7 5 0 LP

Three-color red
laser configuration

143

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Four-color red laser The following map shows the four-color configuration for theconfiguration
640-nm red laser.
PMT

LP mirror

BP filter

Fluorochromes

A

750

780/60APC-Cy7, APC-H7

B

710

730/45Alexa Fluor™ 700

C675

685/35Alexa Fluor™ 680

D—670/14APC

730/45710 LP

7 5 0 LP 675 L

780

/60

P

685

/35

144

67

0/

14

Chapter 9: Detector array configurations

The following map shows the two-color configuration for the
405-nm violet laser.
PMT

LP mirror

BP filter

FluorochromesA

475

525/50

AmCyan, Qdot® 525,
BD Horizon™ V500-C,
BD Horizon™ Brilliant Violet™
BV510

B

Blank

450/50

Pacific Blue™, BD Horizon™ V450,
Marina Blue®, Alexa Fluor™ 405,
BD Horizon™ Brilliant Violet™
BV421

450/50/50525
475 LP

Two-color violet
laser configuration

145

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Three-color violet
laser configuration

The following map shows the three-color configuration for the
405-nm violet laser.
PMT

LP mirror

BP filter

FluorochromesA

595

610/20

Qdot® 605, BD Horizon™
Brilliant Violet™ BV605

B

475

525/50

AmCyan, V500-C, BV510,
Qdot® 525C—

450/50

Pacific Blue™, V450, BV421,
Marina Blue®, Alexa Fluor™ 405

525/50475 LP

/20
450
610
/50
595 LP

146

Chapter 9: Detector array configurations

The following map shows the six-color configuration for the
405-nm violet laser.
PMT

LP mirror

BP filter

Fluorochromes

A

630

655/8

Qdot® 655B

595

610/20

BV605, Qdot® 605

C

575

585/15

Qdot® 585D

545

560/20

Qdot® 565E

475

525/50

AmCyan, V500-C, BV510, Qdot® 525

F

Blank

450/50

Pacific Blue™, V450, BV421,
Marina Blue®, Alexa Fluor™ 405

610/2056

54

5 9 5 LP

0/

20

0
5/5
52
LP
475

450/50P

5L

P

630 LP 575 L

655

/8

585

/15

Six-color violet
laser configuration

147

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Two-color NUV
laser configuration

The following map shows the two-color configuration for the
375-nm near-UV (NUV) laser.
PMT

LP mirror

BP filter

FluorochromesA

505

530/30Indo-1 (Blue)

B

Blank

450/50Indo-1 (Violet), DAPI,
Alexa Fluor™ 350

450/50/30530505 LP

148

Chapter 9: Detector array configurations

Five-color yellowgreen laser
configuration

The following map shows the five-color configuration for the
561-nm yellow-green laser.
PMT

LP mirror

BP filter

Fluorochromes

A

750

780/60PE-Cy7B

685

710/50PerCP-Cy5.5C

635

670/30PE-Cy5D

600

610/20PE-CF594, PE-Texas Red®

E

Blank

586/15PE

710/506160685 LP

0/

20

0L

/60
670
780
/30
750 LP 635L
P

58

6/1

5

P

More information•

About the base configuration (page 133)•

Special order configurations (page 150)

149

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Special order configurations
Introduction

This topic describes some of the more common configurations
available through the special order research program.

Available options

The special order BD LSRFortessa X-20 cell analyzer can be
configured with up to 5 lasers choosing from 11 different special
order wavelength options and a variety of detectors. This section
shows some of the common special order laser/detector
combinations. Contact your local BD Biosciences sales
representative for information about other configurations.

BP filters used with Not all combinations of special order BP filters and lasers are
CS&T
normalized to CS&T settings. In this case, CS&T will generate Qr
and Br numbers that are not comparable from instrument to
instrument. Care should be taken when interpreting the CS&T
reported Qr values on special order BD LSRFortessa X-20
instruments. Please see the latest published filter guides available
on our website (bdbiosciences.com) for more information.
Blue 488 nm (20–100 mW)

The fluorochromes listed in the following table can be used withthis laser wavelength for cell surface markers, live/deaddiscrimination, and cell cycle applications. See Common blue laser
configurations (page 155) for common polygon configuration
maps.
Fluorochromes

BP filters

SSC

488/10

FITC, Alexa Fluor™ 488

530/30PE

575/25575/26576/26585/42

Chapter 9: Detector array configurations

Red 640 nm(40 mW)

Fluorochromes

BP filters

PE-CF594, PE-Texas Red®, PI

605/12
610/20616/23PE-Cy5660/20670/30PerCP, PerCP-Cy5.5, PE-Cy5.5

670/14675/20695/40710/50PE-Cy7780/60The fluorochromes listed in the following table can be used withthis laser wavelength for the cell surface marker application. See
Common red laser configurations (page 157) for two common
polygon configuration maps.
Fluorochromes

BP filters

APC, Alexa Fluor™ 647

670/30Alexa Fluor™ 680

685/35Alexa Fluor™ 700

710/50710/20712/21730/45APC-Cy7, APC-H7

780/60151

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Violet 405 nm(20–100 mW)

The fluorochromes listed in the following table can be used withthis laser wavelength for cell surface marker, live/deaddiscrimination, and cell cycle applications. See Common violet
laser configurations (page 158) for common polygon configuration
maps.
Fluorochromes

BP filters

V450, BV421, Pacific Blue™

450/50450/40450/20440/40

AmCyan, Alexa Fluor™ 430, V500-C,
BV510, DAPI

510/50525/50

Qdot® 525

NA

Qdot® 545

NA

Qdot® 565

560/20560/40

Qdot® 585

585/15585/42

BV605, Qdot® 605

605/12605/40610/20

Qdot® 655

655/8
670/30

BD Horizon™ Brilliant Violet™ BV711,
Qdot® 700, Qdot® 705

710/50712/20

Qdot® 800

780/60800/30Note: If a 561-nm yellow-green laser is configured, a 561 notch
filter is required for Qdot® 565 and Qdot® 585.
Note: The 800/30 bandpass filter is a special order research
program filter.

Chapter 9: Detector array configurations

UV 355 nm (20–100 mW)

Yellow-green 561
nm (50 mW)

The fluorochromes listed in the following table can be used withthis laser wavelength for the cell cycle application with the DAPI
and Hoechst fluorochromes. The wavelength is used for the Ca+2
flux application with both Indo-1 fluorochromes. See Common
UV laser configurations (page 160) for two common polygon
configuration maps.
Fluorochromes

BP filters

DAPI, Hoechst

440/40450/20450/40450/50Indo-1 (Ca Bound)450/50Indo-1 (Ca unbound)

530/30The fluorochromes listed in the following table can be used withthis laser wavelength for cell surface marker and fluorescentprotein applications. See Common yellow-green laser
configuration (page 162) for a common polygon configuration
map.
Fluorochromes

BP filters

PE, DsRed

586/15PE-CF594, PE-Texas Red®,
mCherry

610/20PE-Cy5660/20670/30PE-Cy5.5

710/50PE-Cy7780/60153

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Green 532 nm(150 mW)

The fluorochromes listed in the following table can be used withthis laser wavelength for the cell surface markers application. See
Common green laser configuration (page 163) for a common
polygon configuration map.
Fluorochromes

BP filters

PE575/25PE-CF594, PE-Texas Red®

610/20PE-Cy5660/20670/30PE-Cy5.5

695/40710/50PE-Cy7780/60Chapter 9: Detector array configurations

Common blue laser The following maps show two common configurations for theconfigurations
488-nm blue laser.

PerCP-Cy5.5
PE710/5057

55

6 8 5 LP

5/

26

0L

0
5/5
52
LP
50 5

488/10PP

73 5 LP 6 0 0 L

780

/60

610

/20

FITC

PE-CF594

PE-Cy7SSC

155

BD LSRFortessa X-20 Cell Analyzer User’s Guide

FITC

525/505 0 5 LP

/50
488
710
/10
685 LP

156

SSC

PerCP-Cy5.5

Note: With the 488-nm blue laser, FITC requires a 525/50 notch
filter if the cytometer also uses a 532-nm laser.

Chapter 9: Detector array configurations

The following maps show two common configurations for the
640-nm red laser.

Alexa Fluor ™ 700

APC

670/30730/45685 LP

/60
670
780
/30
735 LP/60
780
735 LP

Common red laser
configurations

APC-Cy7

APC-Cy7
APC

157

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Common violet
laser
configurations

The following maps show two common configurations for the
405-nm violet laser.

V450 or BV421

450/50/50525
505 LP

158

AmCyan or BV510

Chapter 9: Detector array configurations

BV711
BV605

V500-C or BV510

710/500/20

Qdot® 565

0L

545LP

560/40

P

0

0/5

45

525/502
5/4
58
5
LP 05 LP
57 0

61

60

685 LPP

735 LP 635 L

780

V450 or
BV421

/60

670

/30

Qdot® 585

Qdot® 655

Qdot® 800

Note: If a 561-nm yellow-green laser is configured, a 561 notch
filter is required for Qdot® 565 and Qdot® 585.

159

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Common UV laser
configurations

The following maps show two common configurations for the355-nm UV laser.

Indo-1 (violet)

450/50/50525
505 LP

160

Indo-1 (blue)

Chapter 9: Detector array configurations

Indo-1 (blue)

525/505 0 5 LP

/50
450
670
/50
630 LP

Indo-1 (violet)

Hoechst Red

161

BD LSRFortessa X-20 Cell Analyzer User’s Guide

The following map shows a common configuration for the 561-nm
yellow-green laser.

PE-Cy5.5
PE-CF594

710/5061

0/60

6 8 5 LP

20

P

0L

5
6/1
58
LP
570

Common yellowgreen laser
configuration

/60
660
780
/20
735 LP 630 L
P

162

PE

PE-Cy5PE-Cy7

Note: When you use a PE conjugate with a 561-nm laser, you must
replace the 585/42 bandpass filter (BP) with a 582/15 BP,
regardless of which laser (blue, green, or yellow-green) is exciting
the PE fluorochromes.

Chapter 9: Detector array configurations

The following map shows a common configuration for the 532-nm
green laser.

PE-Cy5.5
PE-CF594

710/50 or
695/4061
6 8 5 LP
0/

60

20

58

5/4

2

P

0L
/60
660
780
/20
735 LP 630 L
P

Common green
laser configurationPE

PE-Cy5PE-Cy7163

This page intentionally left blank10
Manual settings
This chapter covers the following topics:•

About laser delay (page 166)•

Optimizing laser delay (page 167)•

Adjusting area scaling (page 169)

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

About laser delay
Introduction

This topic describes how to manually set the laser delay if you are
not using CS&T for cytometer setup and tracking.

About laser signal
delay

Sample interrogation takes place within the cuvette flow cell. Laserlight is directed through a series of prisms that focus multiple lasers
on the event stream at different positions. This allows optimal
detection of fluorescent signals from each laser with minimal crosscontamination from the other beams.
For example, in a BD LSRFortessa X-20 four-laser system, the blue
laser intercepts the stream first, followed by the violet, UV, and redlasers. Because the laser signals are spatially separated, there is aslight delay between the detection of each laser’s signal.
Event intercepts yellow-green laser*
Event intercepts red laser

Yellow-green

Event intercepts UV laser
Red

Event intercepts violet laser
Event intercepts blue laser

UV

VioletBlue
Time*The yellow-green laser is only available through the BD special order research program.

The laser delay setting in BD FACSDiva software is used to re-alignthe signals so they can be measured and displayed on the same timescale. Signals are aligned with respect to the blue laser, so the blue
laser will have a 0 delay value, and the red laser will have thelongest delay.

Chapter 10: Manual settings

Optimizing laser delay
Introduction

This topic describes how to optimize the laser delay using
BD FACSDiva software.

Before you begin

To optimize the delay for a given laser, you must acquire eventsfrom a sample with a fluorescence signal excited by that laser.
Follow the procedures in Recording and analyzing data (page 85),for sample optimization and acquiring data.

Procedure

To optimize laser delay:
1. While acquiring data from your sample, create a histogram toshow the fluorescence signal excited by the laser for which thedelay is to be optimized.
2. In the Acquisition Dashboard, set the Events to Display to 500
events.
3. Click the Laser tab in the Cytometer window.
Window extension and laser delay values are displayed in
microseconds (µs).

167

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

4. Set the Window Extension value to 0 µs.
5. Set an initial laser delay value only for the laser you are
optimizing.
• If you are optimizing the violet laser, set its delay to 40 µs.
• If you are optimizing the UV laser, set its delay to 75 µs.
• If you are optimizing the red laser, set its delay to 110 µs.
6. While observing the positive events on the histogram, adjust
the laser delay in 1-µs increments. You might need to adjust
the delay above or below the initial setting.
Choose the setting that moves the events farthest to the right(highest fluorescence intensity).
7. Draw an interval gate on the histogram for the positive events.
8. Create a statistics view to display the mean fluorescenceintensity (MFI) of the gated population.
9. While observing the MFI for the gated population, adjust thelaser delay in 0.1-µs increments within a range of 2.0 µs of thesetting obtained in step 6.
Preserve the setting that maximizes the fluorescence intensity.
10. Reset the Window Extension to 10 µs.

Chapter 10: Manual settings

Adjusting area scaling
Introduction

This topic describes how to manually adjust the area scaling on
your cytometer if necessary for your application. The area scaling
is automatically set in CS&T. Depending on the size of your target
particle, you might need to adjust the area scaling manually. Larger
particles are more likely to require an area scaling adjustment.

About area scaling

The area of a pulse is calculated by BD FACSDiva using measured
height and width measurements. It is sometimes important toverify that the area calculation and the height measurement areequivalent by adjusting the factor applied to the area. The required
area scaling factor changes based on sheath pressure and particle
size.

About this example The following example describes how to adjust area scaling for an
experiment which uses only the 488-nm laser and the 640-nm
laser. You must adjust area scaling for all lasers used in yourexperiment. To adjust the other lasers, add a parameter and thecorresponding plots from that laser to the procedure.
Procedure

To adjust area scaling:
1. Open an existing experiment, or create a new experiment in
the Browser.
2. Create a new specimen by clicking the New Specimen buttonon the Browser toolbar.

169

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

3. In the Inspector, click the Parameters tab and select the H
checkbox to select height for each parameter.

4. On the global worksheet, create the following plots andhistograms:• FSC vs SSC dot plot
• FSC-H and FSC-A histogram• FITC-H and FITC-A histogram• APC-H and APC-A histogram
5. Create a P1 gate in the FSC vs SSC plot, and show only the P1
population in all histograms.

Chapter 10: Manual settings

6. Create three statistic views showing the following:• FSC-H and FSC-A means for P1• FITC-H and FITC-A means for P1• APC-H and APC-A means for P1
Your worksheet should look similar to the following figure.

7. Expand the new specimen, then set the current tube pointer totube_001.

171

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

8. Install the FITC-positive control tube onto the loading port
and click Load in the Acquisition Dashboard.
9. Adjust the FSC and SSC voltages to place the particles onscale.

10. Adjust the P1 gate around the population of interest.
11. Adjust the FSC area scaling.
a. Click the Laser Tab in the Cytometer window.
b. Adjust the FSC area scaling factor until the FSC-A signal
matches the FSC-H signal:• Increase the area scaling factor if the FSC-A signal islower than FSC-H.

Chapter 10: Manual settings• Decrease the area scaling factor if the FSC-A signal ishigher than FSC-H.

c. View the result of your change in the histograms andstatistics views.
12. Adjust the blue laser area scaling factor until the FITC-A signal
matches the FITC-H signal, if needed.
13. Unload the FITC-positive control tube, then load the APCpositive control tube.

173

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

14. Adjust the red laser area scaling factor until the APC-A signal
matches the APC-H signal, if needed.

11
Supplies and consumables
This chapter covers the following topics:•

Ordering information (page 176)•

Beads (page 176)•

Reagents (page 177)•

Equipment (page 178)

176

BD LSRFortessa X-20 Cell Analyzer User’s Guide

Ordering information
To order spare parts and consumables from BD Biosciences:•

Within the US, call (877) 232-8995.

•

Outside the US, contact your local BD Biosciences customer
support representative.

Worldwide contact information can be found at
bdbiosciences.com.

Beads
Introduction

This topic lists the QC and CS&T beads available.

QC particles
Particle

Laser

Supplier

Catalog No.

SPHERO™ Rainbow Calibration
Particles (8 peaks)All

BD Biosciences

559123SPHERO Ultra Rainbow
Fluorescent Particles (single peak)All

Spherotech, Inc.

URFP-30-2

BD DNA QC Particles

Blue (488 nm)

BD Biosciences

349523

Chapter 11: Supplies and consumables

CS&T beads
Bead

Laser

BD FACSDiva
CS&T research
beads



UV (355 nm and 375 nm)

Violet (405 nm)

Blue (488 nm)

Red (640 nm)

Yellow-green (561 nm)

Green (532 nm)

Supplier

Catalog No.

BD Biosciences



655050(50 tests)

655051(150 tests)

Reagents
Reagent

Supplier

Catalog No.

BD FACSFlow sheath fluid

BD Biosciences

342003

BD FACS™ sheath solution
with surfactant (recommended
for use with the HTS option)

BD Biosciences

336524

Monoclonal antibodies

BD Biosciences

See the BD Biosciences
Product Catalog or the
BD Biosciences website(bdbiosciences.com)

BD FACS™ lysing solution

BD Biosciences

349202

BD Detergent Solution
Concentrate

BD Biosciences

660585177

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

Reagent

Supplier

Catalog No.

BD FACSClean solution

BD Biosciences

Dyes and fluorochromes

BD Biosciences, Life
Technologies, or Sigma–

Chlorine bleach(5% sodium hypochlorite)

Clorox® or other major
supplier (to ensure that thebleach is at the correct
concentration and free ofparticulate matter)–

340345Equipment
Equipment item

Supplier

Bal seal

BD Biosciences

Catalog No.
343509O-ring, sample tube

343615

Sheath filter assembly

345734

Falcon polystyrene test tubes, 12 x 75-mm

Corning

352008

IndexNumerics

B355-nm UV laser 153, 160
375-nm near-UV laser 148
405-nm violet laser 145–147, 152, 158
488-nm blue laser 139–141, 150, 155532-nm green laser 154, 163
561-nm yellow-green laser 153, 162
640-nm red laser 142–144, 151, 157

Bal seal
ordering 178
replacing 58
bandpass (BP) filters
detector array placement 25
theory 107
base configurations 137
batterychanging 53
test 52
BD Detergent Solution Concentrate 48
BD FACSClean solution 178
BD FACSDiva software See software
BD FACSFlow sheath fluid 177
BD FACSFlow solution 33
BD FACSFlow supply system 30, 49
BD High Throughput Sampler (HTS) 22
blank optical holders 41
bleach 46, 178
blue 488-nm laser 139–141, 150, 155
BP See bandpass filters
bubbles, removing air 34
buttons
fluid control 20
sample flow rate control 19

Aacridine orange (AO) 47
air bubbles, removing 33
alarm
battery test 52
waste container 23
analysisdata 86
immunophenotyping 91
reusing 97
saving 97
antibodies 102
AO See acridine orange
application settings
applying 87
creating 76
area scaling 169
arm, tube support 21
aspiration (sample), rapid 119
assistance, technical 13Ccalculating compensation 82
capacity, waste container 23

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

cleaning solutions 46
compensation
calculating 82
control tubes 79
recording settings 80
theory 109
components
cytometer shown 17
sheath container shown 32
sheath filter shown 56
waste container shown 37
computer system, about 27
configurationbase 133
BD FACSDiva 66
containers
sheath 23, 31
waste 23, 36, 38
control panel, cytometer 18
controls
fluidics 18
single-stained 65, 86
conventions
safety symbols 11
creating
analysis objects 91
global worksheets 87
statistics view 91
CS&T particles 177
cuvette flow cell 166
CV, troubleshooting 125
cytometer 16
components shown 17
configuration 41, 66
disconnect error 127
door and drawer 42
setup 64
starting 30
troubleshooting 118

D
DAPI, cleaning after using 47
data
analyzing 86, 91
gating 91
recording 86, 88
debris, excessive 124
default configuration 133
delay, laser 166
detector arrays 24
detectors 26
dichroic mirrors
theory 108
digital data 16
digital electronics 114
Diva software See software
DNA, flow rate for analysis 101
droplet containment system 22Eelectronics
digital 114
laser controls 115
pulse 112
pulse measurements 113
threshold 114
troubleshooting 127
emission duration 112
event rate
erratic 123
high 122
low 123
zero 119, 121
excessive debris 124
excitation wavelength 102
experiments
creating 72
immunophenotyping 87
sample optimization 72

Indexspecifying parameters

73

F
FACSFlow sheath fluid 177
FACSFlow solution 33
Falcon tubes 119
filters, optical
bandpass (BP) 25, 107changing 42
dichroic See dichroic mirrors
longpass (LP) 104, 105
notch 105, 156
shortpass (SP) 104, 106
theory 104
filters, sheathattaching new 57
replacing 56
FITC and Stokes shift 103
fixed-alignment lasers 16
flow celldraining 39
fluidics 100
flow rate control buttons 19
fluid control buttons
PRIME 20
RUN 20
STANDBY 20
fluidics
description 19, 100
flow rate control 19
flushing system 50
priming 39
removing air bubbles 34
run mode 20
sample injection port (SIP) 21
fluidics controls, cytometer 18
fluidics interconnect 35
fluorescence
defined 102
emission 112

fluorescence signal, none 122
fluorochromescompensation theory 109
emission spectra 103
measured 41
role in light emission 102
flushing the system 50
forward scatter (FSC) 102
FSC See forward scatterG
gating data 91
global worksheets
analyzing data 91
creating 87
previewing data 86, 97
green 532-nm laser 154, 163

Hhazard symbol definitions 11
Help, accessing 12High Throughput Sampler (HTS)hydrodynamic focusing 100

Iimmunophenotyping
analysis 91
experiment 87
hydrodynamic focusing

101

K
knob, SAMPLE FINE ADJ

L
laser delay
about 166
optimizing 167
lasers

19

22

181

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

options 24
performance check 68
quality control (QC) particles 176
longpass (LP) filters
defined 104
theory 105
LP See longpass filtersMmaintenance
Bal seal 58
battery change 53
battery test 52
cytometer shutdown 49
sample tube O-ring 60
schedule 47
scheduled 50
sheath filter 56
system flush 50
waste management system 52
mirrors, dichroic longpass (LP) filter

Nnotch filters

25

worn

118

P
particles 176, 177
PerCP, Stokes shift 102
performance check 68, 69
photodiode 26
photomultiplier tubes (PMTs) 26
PMT See photomultiplier tubes
polygonlocation 42
shown 25
population hierarchy 91
power switch 17, 18
PRIME, fluid control button 20
priming fluidics system 39
propidium iodide (PI) 47
pulse
definition 112
measurements 113
processors 113

Q105, 156

O
online Help 12
optic holder 41
opticscomponents 23
configuration 66, 67
dichroic mirrors 108
filters 25, 42, 104
location 17
optimization, sample 64
ordering spare parts 176
O-ringordering 178
replacing 60

quality control (QC)
particles 176
troubleshooting 125

Rrecordingcompensation settings 80
compensation tubes 79
data 86, 88
red 640-nm laser 142–144, 151, 157
removing air bubbles, filter 34
replacing
Bal seal 58
battery 53
optical filters 42
sample tube O-ring 60

Indexsheath filter 56
waste air filter 55
reusing analyses 97
RUN
button orange 121
fluid control button 20

S
safety symbol definitions 11
safety symbols 11
sampleoptimization 64
optimization experiment 72
optimization, single-stained
controls 65, 86
SAMPLE FINE ADJ knob 19
sample injection port (SIP)
components 21hydrodynamic focusing 100
location 17
replacing Bal seal 58
replacing sample tube O-ring 60
sample tube
injection 21
not fitting 119
requirements 119
samples, running 88
saving analyses 97
scatterlight 102
parameter distortion 124
setup, cytometer 64
sheathflow 100
pressure 100
sheath container
components shown 32
defined 23
depressurize 32
preparing 32

sheath filter
components shown 56
ordering 178
removing air bubbles 33
replacing 56
sheath fluid 100
backflush 22
BD FACSFlow sheath fluid 177
shortpass (SP) filters 104, 106
side scatter (SSC) 102
signals, amplifying 26
SIP See sample injection port
softwarecytometer control 10
version 27
spare parts, ordering 176
spillover 109
SSC See side scatter
STANDBY, fluid control button 20
starting cytometer 30
statistics views 91
Stokes shift 102
support, technical 13

Ttechnical assistance 13
testing battery 52
thiazole orange (TO) 47
threshold, defined 114
TO See thiazole orange
troubleshooting
cytometer 118
electronics 127
tubes
Falcon 119
requirements 119

Uuser preferences

67

183

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BD LSRFortessa X-20 Cell Analyzer User’s Guide

UV 355-nm laser

153, 160

V
violet 405-nm laser

145–147, 152, 158Wwaste air filter
component shown 37
replacing 55
waste air vent filter
replacing 121
waste container 23
alarm 23
battery test 52
capacity 23
components shown 37
defined 23
emptying 36
pressurized 38, 121
replacing battery 53
window extension 167
workstation, about 27

Yyellow-green 561-nm laser

153, 162