23-11947-01
10/2020
For Research Use Only
Becton, Dickinson and Company
BD Biosciences
2350 Qume Drive
San Jose, CA 95131 USA
bdbiosciences.com
BD Biosciences
European Customer Support
Tel +32.53.720.600
help.biosciences@bd.com
BD LSRFortessa™ X-20
Cell Analyzer
User’s Guide
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, in
any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without
prior written permission from BD
Biosciences.
The information in this guide is subject to change without notice. BD Biosciences reserves the right to change
its products and services at any time to incorporate the latest technological developments. Although this guide
has been prepared with every precaution to ensure accuracy, BD
Biosciences assumes no liability for any errors
or omissions, nor for any damages resulting from the application or use of this information. BD
Biosciences
welcomes customer input on corrections and suggestions for improvement.
Trademarks
BD, the BD Logo, FACS, FACSDiva, FACSFlow, and FACSClean are trademarks of Becton, Dickinson and
Company 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 and
Carnegie Mellon University and are made and sold under license from GE Healthcare only for research and in
vitro 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 for
compliance could void the user’s authority to operate the equipment.
NOTICE: This equipment has been tested and found to comply with the limits for a Class A digital device,
pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against
harmful interference when the equipment is operated in a commercial environment. This equipment generates,
uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction
manual, may cause harmful interference to radio communications. Operation of this equipment in a
residential area is likely to cause harmful interference in which case the user will be required to correct the
interference at his or her own expense. Shielded cables must be used with this unit to ensure compliance with
the Class A FCC limits. This Class A digital apparatus meets all requirements of the Canadian Interference-
Causing Equipment Regulations. Cet appareil numérique de la classe A respecte toutes les exigences du
Réglement sur le matériel brouilleur du Canada.
Compliance information
NOTICE: This laboratory equipment has been tested and found to comply with the EMC and the Low
Voltage Directives. This includes FCC, Part 15 compliance for a Class A Digital Device.
CAUTION: Any unauthorized modifications to this laboratory equipment may affect the Regulatory
Compliance items stated above.
History
Revision Date Change made
23-11947-00 4/2013 Initial release
23-11947-01 10/2020 Removed references of FACSRinse and added BD Detergent
Solution Concentrate in place of FACSRinse.
Contents
Chapter 1: About this guide 9
What 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
BD LSRFortessa X-20 Cell Analyzer User’s Guide6
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 7
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
BD LSRFortessa X-20 Cell Analyzer User’s Guide10
What this guide covers
This guide describes the procedures necessary to operate and
maintain 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 and
operation.
This guide assumes you have a working knowledge of basic
Microsoft® Windows® operation. If you are not familiar with the
Windows operating system, see the documentation provided with
your computer.
Chapter 1: About this guide 11
Conventions
Introduction The following table lists the safety symbols used in this guide to
alert you to potential hazards.
Safety symbols
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.
Symbol Meaning
Caution alert
Identifies a hazard or unsafe practice that could result in
data loss, material damage, minor injury, severe injury, or
death
Biological hazard
Electrical hazard
Laser hazard
BD LSRFortessa X-20 Cell Analyzer User’s Guide12
Help system The help system installed with BD FACSDiva software includes all
content 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 the
help 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 the
BD
FACSDiva Software Help menu (Help > Documentation >
Reference Manual), or by double-clicking the shortcut on the
desktop.
• 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 the
optional automated sheath and waste fluid control system
designed for use with the BD
®
LSR II, BD LSRFortessa™, and
BD
LSRFortessa X-20.
Chapter 1: About this guide 13
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.
This page intentionally left blank
BD LSRFortessa X-20 Cell Analyzer User’s Guide16
Instrument overview
The special order BD LSRFortessa X-20 cell analyzer is an air-
cooled multi-laser benchtop flow cytometer with the ability to
acquire parameters for a large number of colors. It uses
fixed-alignment lasers that transmit light through a flow cell to
configurable polygon detector arrays. These detectors collect and
translate the resulting fluorescence signals into electronic signals.
Cytometer electronics convert these signals into digital data.
Chapter 2: Introduction 17
Components
Introduction This topic describes the instrument’s components.
Instrument
overview
Control panel
Sample injection port (SIP)
Side drawer (polygon
detector arrays)
Power switch (right side)
Heat ventilation slots
Caution! Do not place any objects on top of the
instrument. Blocking the ventilation may cause the
instrument to overheat.
BD LSRFortessa X-20 Cell Analyzer User’s Guide18
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:
• Acquisition status
• System power
More information • Fluidics (page 19)
• Optics (page 23)
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.
HIGHMEDLOWPRIMESTANDBYRUN
SAMPLE
FINE ADJ
ACQUISITION STATUS
SYSTEM POWER
Fluid control buttons Sample flow rate control buttons System indicators
Chapter 2: Introduction 19
Fluidics
Introduction This topic describes the fluidics system.
Purpose The purpose of the fluidics system is to carry the sample out of the
sample tube and into the sensing region of the flow cell. Cells are
carried 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 are
approximately 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 midpoint
value. For example, if the LOW button is pressed, the knob will
give flow rates from approximately 6–24
µL/min.
BD LSRFortessa X-20 Cell Analyzer User’s Guide20
Fluid control Three fluid control buttons (RUN, STANDBY, and PRIME) set the
cytometer 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 to
an 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 the
sample injection port (SIP) and press STANDBY.
•PRIME. Prepares the fluidics system by draining and filling the
flow 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 a
preset time, the flow cell fills with sheath fluid at a controlled
rate 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 21
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 with
an outer droplet containment sleeve. The sleeve works in
conjunction with a vacuum pump to eliminate droplet formation
of sheath fluid as it backflushes from the sample injection tube.
Sample injection tube. Stainless steel tube that carries sample from
the sample tube to the flow cell. This tube is covered with an outer
sleeve that serves as part of the droplet containment system.
Tube support arm. Arm that supports the sample tube and
activates the droplet containment system vacuum. The vacuum is
on when the arm is positioned to the side and off when the arm is
centered.
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.
Outer sleeve
Sample injection tube
Tube support arm
BD LSRFortessa X-20 Cell Analyzer User’s Guide22
Cautions when
using the HTS
option
Droplet
containment
module
The DCM prevents sheath fluid from dripping from the SIP and
provides 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.
Caution: Biohazard! When using the BD LSRFortessa
X-20 cell analyzer with the HTS, ensure that the HTS is
completely pushed into the operating position before
removing the droplet containment module (DCM) sleeve
or 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 the
proper location, the containment dish with padding is
directly below the SIP.
Caution! If you are using the HTS option, always slide
the HTS mount slowly to prevent sample cross-
contamination 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.
Chapter 2: Introduction 23
Sheath and waste containers
Introduction This topic describes the sheath and waste containers. The sheath
and waste containers are outside the cytometer and are positioned
on 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 filtered
through an in-line, interchangeable filter that prevents small
particles from entering the sheath fluid lines.
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)
Optics
Introduction 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
Caution! Do not fill the sheath tank to its maximum
capacity (8 L). When an overfull tank is pressurized,
erratic cytometer performance can result.
BD LSRFortessa X-20 Cell Analyzer User’s Guide24
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.
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 a
variety of wavelengths and powers through our special order
research program.
Side drawer with polygons
Locked cover (lasers
accessible by BD Service only)
Laser Wavelength (nm) Power (mW)
Blue 488 50
Red 640 40
Violet 405 50
NUV 375 50
Yellow-green 561 50
Chapter 2: Introduction 25
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 the
specified 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 with
other wavelengths of filters and mirrors.
Longpass dichroic mirrors
Bandpass filters
BD LSRFortessa X-20 Cell Analyzer User’s Guide26
Signal detectors Light signals are generated as particles pass through the laser beam
in a fluid stream. When these optical signals reach a detector,
electrical pulses are created that are then processed by the
electronics system.
There are two types of signal detectors in the BD LSRFortessa
X-20 cell analyzer:
• Photomultiplier tubes (PMTs). Used to detect the weaker
signals 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 is
available through the BD
special order research program.
More information • Optical filter theory (page 104)
• About the base configuration (page 133)
• Special order configurations (page 150)
Chapter 2: Introduction 27
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:
• Microsoft Windows operating system
•BD FACSDiva software version 7.0 or later for data
acquisition and analysis
• Software documentation including the help system
More information • About the BD LSRFortessa X-20 documentation (page 11)
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)
BD LSRFortessa X-20 Cell Analyzer User’s Guide30
Starting the cytometer and computer
Introduction This topic describes how to start the cytometer and turn on the
computer.
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 to
stabilize.
3. Turn on the computer and log in to Windows.
Note: You can turn on the power to the flow cytometer and
the 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 to
ensure that the cytometer is connected to the workstation.
The cytometer connects automatically. While connecting, the
message Cytometer Connecting is displayed in the status area
of the Cytometer window. When connection completes, the
message changes to Cytometer Connected.
Caution! Failure to warm up and stabilize the
instrument could affect sample data.
Chapter 3: Cytometer setup 31
If the message Cytometer Disconnected appears, see
Electronics 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.
BD LSRFortessa X-20 Cell Analyzer User’s Guide32
Sheath container
components
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 vent
valve.
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.
Air line (green)
Sheath fluid line (blue)
to cytometer
Filter assembly
Tank handle
Cap handle
Vent valve
Clamp knob
Chapter 3: Cytometer setup 33
5. Add 6 L of sheath fluid, such as BD FACSFlow solution, to the
sheath container.
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 is
seated 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 occasionally
dislodge and pass through the flow cell, resulting in inaccurate
data.
Caution! Do not fill the sheath tank to its maximum
capacity (8 L). When an overfull tank is pressurized,
erratic cytometer performance can result.
BD LSRFortessa X-20 Cell Analyzer User’s Guide34
Procedure To remove air bubbles:
1. Check the sheath filter for trapped air bubbles.
2. If bubbles are visible, gently tap the filter body with your
fingers 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.
Vent line
Vent fitting
Cytometer fluid line
(roller clamp not visible)
Chapter 3: Cytometer setup 35
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 a
steady stream of fluid empties from the filter.
4. Tilt the filter and verify that no trapped air remains in the
filter.
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.
Button
Vent fitting
BD LSRFortessa X-20 Cell Analyzer User’s Guide36
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.
When to check the
waste container
Check the fluid levels in the waste container every time you use the
cytometer. This ensures that the waste container does not become
too full.
Caution: Biohazard! All biological specimens and
materials coming into contact with them are considered
biohazardous. Handle as if capable of transmitting
disease. Dispose of waste using proper precautions and in
accordance with local regulations. Never pipette by
mouth. Wear suitable protective clothing, eyewear, and
gloves.
Chapter 3: Cytometer setup 37
Waste container
components
Cautions
Waste tubing (orange) from
cytometer
Level sensor line
Bracket
Waste air vent filter
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).
BD LSRFortessa X-20 Cell Analyzer User’s Guide38
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 to
empty it.
3. Empty the waste container.
4. Add approximately 1 L of bleach to the waste container and
close it.
5. Reconnect the orange waste tubing and make sure it is not
kinked.
6. Reconnect the level sensor line.
Caution! If the air vent filter on the top of the waste
container cap assembly becomes clogged, air cannot be
vented from the container, causing it to swell under
pressure. 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 the
waste container cap assembly without first relieving the
pressure might generate an unnoticeable aerosol. Use
appropriate precautions when troubleshooting a clogged
air filter. Wear suitable protective clothing, eyewear, and
gloves.
Caution! The waste container is heavy when full.
When emptying it, use good body mechanics to
prevent injury.
Chapter 3: Cytometer setup 39
Priming the fluidics
Introduction This topic describes how to prime the fluidics system.
When to prime the
fluidics
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 is
necessary 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 cytometer
in standby mode.
More information • Cytometer troubleshooting (page 118)
BD LSRFortessa X-20 Cell Analyzer User’s Guide40
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 are
labeled 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 is
marked accordingly.
The filters steer progressively shorter wavelengths of light to the
next PMT in the array as indicated by the lines and arrows on the
top of the polygon.
PMT A
Optic holder
handle
Optic holders
Chapter 3: Cytometer setup 41
Optic holders,
filters, and mirrors
Optic holders house filters and mirrors. Your cytometer includes
several blank (empty) optic holders.
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 your
field service engineer.
BD FACSDiva
cytometer
configuration
Before you acquire data using BD FACSDiva software, you must
specify 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 your
BD
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)
Caution! To ensure data integrity, do not leave any slots
empty in a detector array when you are using the
associated laser. Always use a blank optic holder.
BD LSRFortessa X-20 Cell Analyzer User’s Guide42
Changing optical filters and mirrors
Introduction This topic describes how to verify that the optical filters are in the
appropriate position for your particular requirements. Before you
run samples, you must set up the optical filters.
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 is
in the last position used.
4. Close the cytometer side drawer.
More information • Detector array configurations (page 129)
Caution: Laser Hazard! Follow the precautions outlined in
the BD LSRFortessa X-20 Safety and Limitations Guide
while changing optical filters or mirrors.
Chapter 3: Cytometer setup 43
Custom configurations and baselines
Introduction This topic describes where to find information on how to create a
custom configuration and define a baseline for a performance
check.
Overview BD Cytometer Setup and Tracking (CS&T) software is used to
define 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 this
case, CS&T will generate sensitivity and background values (Qr
and 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 a
baseline.
More information • Running a performance check (page 68)
This page intentionally left blank
4
Maintenance
This 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)
BD LSRFortessa X-20 Cell Analyzer User’s Guide46
Maintenance overview
Introduction This topic provides an overview of the special order
BD
LSRFortessa X-20 cell analyzer routine maintenance and
cleaning procedures.
General use
guidelines
Follow these guidelines whenever operating or maintaining the
cytometer:
• Handle all biological specimens and materials as if they are
capable of transmitting disease.
• Dispose of waste using proper precautions and in accordance
with 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 to
appropriate concentrations.
Caution: Biohazard! Coming into contact with all
biological specimens and materials is considered
biohazardous.
Caution! Higher concentrations of sodium hypochlorite
and use of other cleaning solutions might damage the
cytometer.
Chapter 4: Maintenance 47
When to perform
maintenance
procedures
Perform maintenance procedures in the following frequencies.
Cleaning the fluidics
Introduction This topic describes how to perform the daily fluidics cleaning.
Overview Cleaning the fluidics daily prevents the sample injection tube from
becoming 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.
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 the
cytometer)
Changing the sheath filter (page 57)
Changing the Bal seal (page 59)
Changing the sample tube O-ring (page 61)
BD LSRFortessa X-20 Cell Analyzer User’s Guide48
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) and
allow the cleaning solution to run for 5 minutes with the
sample 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.
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 to
prevent salt deposits from forming in the injection tube. This
tube also catches back drips from the flow cell.
Caution! Do not mix BD Detergent Solution Concentrate
and bleach because they produce chlorine gas.
Caution! Do not leave more than 1 mL of water on
the SIP. When the instrument is turned off or left in
standby mode, a small amount of fluid will drip back
into the sample tube. If there is too much fluid in the
tube, it could overflow and affect the cytometer
performance.
Chapter 4: Maintenance 49
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 a
system flush and leave the fluidics system filled with DI water to
prevent saline crystals from clogging the fluidics.
More information • Cleaning the fluidics (page 47)
• Flushing the system (page 50)
BD LSRFortessa X-20 Cell Analyzer User’s Guide50
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 the
BD
FACSFlow Supply System User’s Guide for instructions on
flushing the system.
Cautions
Procedure 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.
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.
Caution: Biohazard! The cytometer hardware might be
contaminated with biohazardous material. Use 10%
bleach to decontaminate the instrument.
Caution! Do not run detergent, bleach, or ethanol
through the sheath filter. They can break down the
filter paper within the filter body, causing particles to
escape into the sheath fluid, possibly clogging the
flow cell.
Chapter 4: Maintenance 51
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 the
sheath 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.
14. Repeat steps 2 through 11 with DI water.
15. Replace the sheath filter and refill the sheath container with
sheath fluid.
Caution! Do not mix BD Detergent Solution Concentrate
and bleach because they produce chlorine gas.
Chapter 4: Maintenance 53
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 perform
the 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.
2. Toggle the switch.
If the battery and the alarm are working properly, you should
hear an alarm buzzing. If you do not hear any sound, change
the battery as described in the following section.
3. Release the switch.
BATTERY TEST
Battery test
BD LSRFortessa X-20 Cell Analyzer User’s Guide54
Changing the
battery
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 55
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.
BD LSRFortessa X-20 Cell Analyzer User’s Guide56
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
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.
Caution: Biohazard! Treat a contaminated air filter as
biohazardous waste.
Chapter 4: Maintenance 57
Changing the sheath filter
Introduction This topic describes how to change the sheath filter. The sheath
filter 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 and
consumables (page 175) for ordering information.
Sheath filter
components
Filter base
Quick-disconnect
Sheath line
Vent fitting
Vent line
BD LSRFortessa X-20 Cell Analyzer User’s Guide58
Removing the old
filter
To remove the old filter:
1. Place the cytometer in standby mode.
2. Remove the sheath filter assembly by pressing the
quick-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 new
filter
To 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 the
filter to the filter base.
3. Connect the sheath line to the filter assembly by squeezing the
quick-disconnect.
4. Attach the cytometer fluid line to the filter assembly via the
quick-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 a
steady stream of fluid empties from the filter.
6. Tilt the filter and verify that no trapped air remains in the
filter.
7. Repeat steps 5 and 6 as necessary to remove all trapped air.
Chapter 4: Maintenance 59
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 with
the sample tube and ensures proper tube pressurization.
When to change
the Bal seal
Over time, the Bal seal becomes worn or cracked and requires
replacement. Replacement is necessary if a proper seal is not
formed when a sample tube is installed on the SIP. Indications that
a 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 the
cytometer, the RUN button is orange (not green).
Procedure To replace the Bal seal:
1. Remove the outer sleeve from the sample injection tube by
turning the retainer counter-clockwise. Slide the outer sleeve
down and off of the sample injection tube.
Work carefully. The outer sleeve can fall off as you loosen the
retainer.
Retainer
Outer sleeve
Sample injection tube
Bal seal
BD LSRFortessa X-20 Cell Analyzer User’s Guide60
2. Remove the Bal seal by gripping it between your thumb and
index 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 the
retainer.
4. Re-install the retainer and outer sleeve over the sample
injection tube. Push the outer sleeve all the way up into the
sample 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 the
retainer.
Chapter 4: Maintenance 61
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 seal
that allows the droplet containment vacuum to function properly.
When to replace
the O-ring
Replace the O-ring when droplets form at the end of the sample
injection tube while the vacuum is operating.
Caution
Procedure To change the O-ring:
1. Remove the outer sleeve from the sample injection tube by
turning the retainer counter-clockwise.
2. Slide the outer sleeve from the retainer.
Caution: Biohazard! Cytometer hardware might be
contaminated with biohazardous material. Wear suitable
protective clothing, eyewear, and gloves whenever
cleaning the cytometer or replacing parts.
Retainer
O-ring
This page intentionally left blank
3. Invert the outer droplet sleeve and allow the O-ring to
fall onto the benchtop.
If the O-ring does not fall out initially, hold the O-ring
with your free hand and slide the outer sleeve to remove
the O-ring.
4. Place the new O-ring into the retainer. Make sure the
O-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 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 the retainer.
Chapter 4: Maintenance 63
BD LSRFortessa X-20 Cell Analyzer User’s Guide64
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)
BD LSRFortessa X-20 Cell Analyzer User’s Guide64
Cytometer settings workflow
Introduction This topic describes how to optimize cytometer settings. The
optimization is performed using the Cytometer Setup and
Tracking, Application Settings, and Compensation Setup features
of BD
FACSDiva software.
When to optimize
settings
Before you record data for a sample, optimize the cytometer
settings for the sample type and fluorochromes used.
Manual
compensation
Compensation setup automatically calculates compensation
settings. If you choose to perform compensation manually, not all
of 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 first
time, you should be familiar with BD
FACSDiva software
concepts: 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 and
perform the setup and QC procedures. See
Cytometer setup
(page 29).
Workflow for
optimizing settings
Cytometer optimization consists of the following steps.
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 65
Note: Application settings are optional and do not have to be
saved for the experiments. However, they are useful for optimizing
cytometer settings.
About the
examples
The examples in this chapter use a 4-color bead sample with the
following fluorochromes:
•FITC
•PE
• PerCP-Cy™5.5
•APC
If you follow this workflow with a different bead sample (or
another sample type), your software views, data plots, and
statistics might differ from the example. Additionally, you might
need 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.
4 Creating application settings (page 76)
5 Recording compensation controls (page 79)
6 Calculating compensation (page 82)
Step Description
BD LSRFortessa X-20 Cell Analyzer User’s Guide66
Verifying the configuration and user preferences
Introduction This topic describes how to verify the cytometer configuration and
user preferences before you create an experiment.
Procedure To verify the configuration and preferences before you create an
experiment:
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 the
following parameters: FITC, PE, PerCP-Cy5.5, and APC.
Caution! To obtain accurate data results, the current
cytometer configuration must reflect your
BD LSRFortessa X-20 cell analyzer optics.
Chapter 5: Optimizing cytometer settings 67
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 your
BD
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)
BD LSRFortessa X-20 Cell Analyzer User’s Guide68
Running a performance check
Introduction This topic describes how to run a performance check as part of
quality control.
Overview The CS&T application is designed to monitor performance on a
daily basis and to optimize laser delay.
Running a performance check on a regular basis provides a
standard for monitoring changes in performance due to
degradation of laser power, aging of PMTs, and other potential
cytometer service issues. Performance results are also affected by
fluidics performance. We strongly recommend following the
fluidics maintenance procedures as described in
Cleaning the
fluidics (page 47).
Considerations Some BP filters might not be normalized to CS&T settings. In this
case, CS&T will generate Qr and Br numbers that are not
comparable 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 non-
CS&T normalized filter.
Before you begin Define the performance baseline for any configuration before
running a performance check. See
Custom configurations and
baselines (page 43).
Chapter 5: Optimizing cytometer settings 69
Procedure To 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 your
experiment.
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).
BD LSRFortessa X-20 Cell Analyzer User’s Guide70
6. Install the bead tube onto the SIP.
7. In the Setup Control window, select Check Performance from
the 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 is
run. The performance check takes approximately 5 minutes to
complete.
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 should
have a green checkbox displayed and the word Passed next to
it.
If any parameters did not pass, see the BD Cytometer Setup
and Tracking Application Guide for troubleshooting
information.
Chapter 5: Optimizing cytometer settings 71
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, and
other cytometer settings will be updated to the latest settings
from 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).
BD LSRFortessa X-20 Cell Analyzer User’s Guide72
Setting up an experiment
Introduction This topic describes how to create an experiment in a new folder,
specify the parameters of the experiment, and add compensation
tubes.
Creating an
experiment
To create an experiment:
1. Click the buttons on the Workspace toolbar to display the
following windows as needed:
•Browser
• Cytometer
• Inspector
• Worksheet
• Acquisition Dashboard
When you add elements or make selections in the Browser, the
Inspector 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 73
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.
BD LSRFortessa X-20 Cell Analyzer User’s Guide74
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 75
a. Click the selection button (to the left of the parameter
name) to select the parameter.
b. Click Delete.
The parameter is deleted.
BD LSRFortessa X-20 Cell Analyzer User’s Guide76
Creating application settings
Introduction This topic describes how to create application settings.
About application
settings
Application settings are associated with a cytometer configuration
and include the parameters for the application, area scaling values,
PMT voltages, and threshold values, but not compensation. Each
time a performance check is run for a configuration, the
application 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 performance
check 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 created
according to the selections in the Parameters tab.
Chapter 5: Optimizing cytometer settings 77
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 the
application.
• Optimize the FSC and SSC voltages to place the population
of 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 the
center of the negative population with the crosshair visible
in the gray box.
Note: Do not decrease the fluorescence PMT voltages. Doing
so can make it difficult to resolve dim populations from the
negative population.
4. Unload the unstained control tube from the cytometer.
5. Load the multicolor sample onto the cytometer or load single-
color control tubes and verify each fluorochrome signal
separately.
BD LSRFortessa X-20 Cell Analyzer User’s Guide78
6. Verify that the positive populations are on scale.
If a positive population is off scale, lower the PMT voltage for
that 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 the
cytometer 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 79
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-generic
tube labels.
2. Click OK.
BD LSRFortessa X-20 Cell Analyzer User’s Guide80
Compensation control tubes are added to the experiment.
Worksheets containing appropriate plots and gates are added
for each compensation tube.
Recording
compensation
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 (it
becomes 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 81
Since the application settings have been optimized for your
sample, the cytometer settings should not require adjustment
other than the changing of FSC and SSC voltages to place the
beads 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 with
your changes.
8. Click Record Data.
9. When recording is finished, remove the unstained control tube
from the cytometer.
10. Click Next Tube.
11. Install the next tube onto the cytometer and repeat steps 8
through 10 until data for all stained control tubes has been
recorded.
12. Double-click the first stained control tube to display the
corresponding worksheet.
Caution! Do not change the PMT voltages after the
first compensation control has been recorded. In
order to calculate compensation, all controls must
be recorded with the same PMT voltage settings. If
you need to adjust the PMT voltage for a
subsequent compensation control, you must record
all compensation controls again.
BD LSRFortessa X-20 Cell Analyzer User’s Guide82
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 the
data 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 to
enter a name for the compensation setup. The default name is
year/month/day/time.
Chapter 5: Optimizing cytometer settings 83
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 experiment
name, date, or both in the setup name.
The compensation setup is linked to the MyExperiment
cytometer 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 the
positive controls should be aligned with the means of the negative
controls.
More information • Recording compensation controls (page 79)
This page intentionally left blank
BD LSRFortessa X-20 Cell Analyzer User’s Guide86
Data recording and analysis workflow
Introduction This topic outlines the basic acquisition and analysis tasks using
BD
FACSDiva software.
About the
examples
The 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 the
procedure.
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 for
recording and
analyzing data
Recording and analyzing data consists of the following steps.
Step Description
1 Preparing the workspace (page 87)
2 Recording data (page 88)
3 Analyzing data (page 91)
4 Reusing an analysis (page 97)
Chapter 6: Recording and analyzing data 87
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 to
access the default global worksheet, and rename the worksheet
MyData.
4. On the MyData worksheet, create the following plots for
previewing the data:
• FSC vs SSC
•FITC vs PE
• FITC vs PerCP-Cy5.5
•FITC vs APC
Applying saved
application settings
to a new
experiment
When applications settings are applied to an experiment, the
cytometer settings are updated with the parameters included in the
application 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 select
Application Settings > Apply.
BD LSRFortessa X-20 Cell Analyzer User’s Guide88
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 the
cytometer 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 89
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 the
singlet 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.
BD LSRFortessa X-20 Cell Analyzer User’s Guide90
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 8
through 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 91
Analyzing data
Introduction This topic describes how to analyze recorded tubes by creating
plots, 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.
BD LSRFortessa X-20 Cell Analyzer User’s Guide92
7. Select all plots except the FSC vs SSC plot, and use the Plot tab
in 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 93
9. Select the Tube and Populations checkboxes to display their
names 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 tab
of the Inspector.
BD LSRFortessa X-20 Cell Analyzer User’s Guide94
• 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 APC-
positive population. Name the population APC positive in
the 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 and
Tube 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 the
fluorescence parameters.
Chapter 6: Recording and analyzing data 95
e. Click OK.
12. Print the analysis.
BD LSRFortessa X-20 Cell Analyzer User’s Guide96
Your global worksheet analysis objects should look like the
following.
Chapter 6: Recording and analyzing data 97
More information • Reusing an analysis (page 97)
Reusing an analysis
Introduction This topic describes how to use a global worksheets to apply the
same analysis to a series of recorded tubes. Once you define an
analysis for a tube, you can use it to analyze the remaining tubes in
the 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 the
worksheet. To avoid altering a global worksheet, save an
analysis to a normal worksheet, then make adjustments on the
normal 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 after
recording 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.
BD LSRFortessa X-20 Cell Analyzer User’s Guide98
2. Right-click its analysis and select Copy.
3. Click the Worksheets View button on the Worksheet toolbar to
switch 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 select
Paste.
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 to
view the analysis.
Applying an
analysis to normal
worksheets
You can apply the global worksheet analysis to multiple tubes (on
a 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)
BD LSRFortessa X-20 Cell Analyzer User’s Guide100
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 is
pressure-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 slight
overpressure relative to the sheath fluid. The conical shape of the
lower chamber creates a laminar sheath flow that carries the
sample core upward through the center of the flow cell, where the
particles to be measured are intercepted by the laser beam. This
process is known as hydrodynamic focusing.
The objective in flow cytometric analysis is to have at most one cell
or 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.
Low sample
pressure
(12 µL/min)
High sample
pressure
(60 µL/min)
Laser beam Laser beam
Sheath
fluid
Sheath
fluid
Sheath
fluid
Sheath
fluid
Sample Sample
Chapter 7: Technical overview 101
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 less
resolved, 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 to
intercept the laser beam properly. Always ensure that the fluidics
system is free of air bubbles and debris, and is properly
pressurized.
About optics
Introduction 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)
Optics system The optics system consists of lasers, optical filters, and detectors.
Lasers illuminate the cells or particles in the sample and optical
filters direct the resulting light scatter and fluorescence signals to
the appropriate detectors.
BD LSRFortessa X-20 Cell Analyzer User’s Guide102
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 the
laser beam is called forward scatter (FSC) and light that scatters
perpendicular to the laser beam is called side scatter (SSC).
FSC and SSC are related to certain physical properties of cells.
•FSC. Indicates relative differences in the size of the cells or
particles. Larger cells scatter more light and therefore they are
higher 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.
Fluorescence When cells or particles stained with fluorochrome-conjugated
antibodies or other dyes pass through a laser beam, the dyes can
absorb 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) and
emitting red light (675
nm), while other fluorochromes such as
Light source
Side scatter (SSC)
Forward scatter (FSC)
Chapter 7: Technical overview 103
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 some
commonly 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.
BD LSRFortessa X-20 Cell Analyzer User’s Guide104
Optical filter theory Optical filters modify the spectral distribution of light scatter and
fluorescence directed to the detectors. When photons encounter an
optical filter, they are either transmitted, absorbed, or reflected.
Even though an optical filter is rated at its 50% transmission point,
the filter passes (lets through) a minimal amount of light outside of
this 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-of-
band wavelengths. A less steep slope indicates that more light
outside the rated bandwidth is being transmitted.
Types of optical
filters
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 not
recommended, 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.
Photons
absorbed
Photons
reflected
Photons
transmitted
Chapter 7: Technical overview 105
• 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.
Longpass (LP)
filters
LP filters pass wavelengths longer than the filter rating. For
example, a 500-LP filter permits wavelengths 500 nm or longer to
pass through it and either absorbs or reflects wavelengths shorter
than 500 nm.
Longpass
Wavelength (nm)
% Transmission
BD LSRFortessa X-20 Cell Analyzer User’s Guide106
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
Wavelength (nm)
% Transmission
Chapter 7: Technical overview 107
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 width
of the band of light that is passed. For example, a 500/50-BP filter
transmits 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.
The performance of an optical BP filter depends on the optical
transmission. Sample transmission curves are shown in the
following 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 optical
coatings and unique manufacturing processes. For more
demanding multicolor applications, higher performance filters are
available through the special order research program. Contact
your local BD Biosciences sales representative for details.
Bandpass
Wavelength (nm)
% Transmission
BD LSRFortessa X-20 Cell Analyzer User’s Guide108
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 to
dichroic 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 have
a surface coating that reflects certain wavelengths, but many LP or
SP filters are absorbance filters that do not have any specific
reflective characteristics. Also, optical filters and dichroic mirrors
are rated at a specific angle of incidence. When used in front of the
fluorescence 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.
Wavelength (nm)
% Transmission
Chapter 7: Technical overview 109
Compensation
theory
Fluorochromes emit light over a range of wavelengths. Optical
filters are used to limit the range of frequencies measured by a
given detector. However, when two or more fluorochromes are
used, the overlap in wavelength ranges often makes it impossible
for optical filters to isolate light from a given fluorochrome. As a
result, light emitted from one fluorochrome appears in a detector
intended for another. This is referred to as spillover. Spillover can
be 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 excitation
of fluorochromes. In the following example, excitation with a 561-
nm laser (special order) will help minimize spillover.
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.
Wavelength (nm)
Normalized intensity
BD LSRFortessa X-20 Cell Analyzer User’s Guide110
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 and
negative FITC populations have the same means in the PE
parameter statistics.
The following image shows the FITC spillover optimally
compensated out of the PE parameter.
Once fluorescence compensation has been set for any sample, the
compensation setting remains valid for a subsequent dim or bright
sample (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.
PE
FITC
FITC-positive
population
Unstained
particles
PE
FITC
Unstained
particles
FITC-positive
population
Matching means
Chapter 7: Technical overview 111
FITC PE
Different intensity FITC signals
Same proportion or percentage of
spectral overlap in PE channel
Wavelength (nm)
Normalized intensity
BD LSRFortessa X-20 Cell Analyzer User’s Guide112
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, they
scatter the laser light and can emit fluorescence. Because the laser
beam is focused on a small spot and particles move rapidly through
the flow cell, the scatter or fluorescence emission signal has a very
brief duration—only a few microseconds. This brief flash of light is
converted into an electrical signal by the detectors. The electrical
signal is called a pulse. The following figures illustrate the anatomy
of a pulse.
1. A pulse begins when a particle enters the laser beam. At this
point, both the beam intensity and signal intensity are low.
2. The pulse reaches a maximum intensity or height when the
particle reaches the middle of the beam, where the beam and
signal intensity are the brightest. The peak intensity, or height
of the pulse, is measured at this point.
Time
Signal intensity
Time
Signal intensity
Chapter 7: Technical overview 113
3. As the particle leaves the beam, the pulse trails off below the
threshold.
Pulse
measurements
The pulse processors measure pulses by three characteristics:
height, area, and width.
• Height. The maximum digitized intensity measured for the
pulse.
• 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 the
window extension value added after the pulse drops below the
threshold. The window gate extends until the pulse is 75% of
the initial threshold.
• Width. Calculates:
Time
Signal intensity
Area
Height
x 64,000
BD LSRFortessa X-20 Cell Analyzer User’s Guide114
Digital electronics BD LSRFortessa X-20 electronics digitize the signal intensity
produced by a detector. The digitized data is stored in memory and
further 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 measure
signal pulses. A threshold is defined for a specific detector signal.
The system continuously samples the digitized signal data and
calculates 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 threshold
value (AND threshold in the software)
• Intervals during which ANY signal exceeds its threshold value
(OR threshold in the software)
Chapter 7: Technical overview 115
Laser controls Controls in the Laser tab of the Cytometer window are used to
manually 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)
This page intentionally left blank
BD LSRFortessa X-20 Cell Analyzer User’s Guide118
Cytometer troubleshooting
Introduction This topic describes possible problems and recommended solutions
for special order BD
LSRFortessa X-20 cell analyzer issues.
Droplets are visible
on the SIP
Possible causes Recommended solutions
Worn O-ring in the retainer Replace the O-ring. See Changing the
sample tube O-ring (page 60).
Outer sleeve is not seated in
the retainer
1. Loosen the retainer.
2. Push the outer sleeve up into the
retainer until seated.
3. Tighten the retainer.
Outer sleeve is not on the
sample injection tube
Replace the outer sleeve.
1. Loosen the retainer.
2. Slide the outer sleeve over the
sample injection tube until it is
seated.
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 High
Throughput Sampler User’s Guide for
more information.
Chapter 8: Troubleshooting 119
Sample tube not
fitting on SIP
Rapid sample
aspiration
No events in
acquisition 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 are
visible on the SIP (page 118). If the
issue is not resolved, call your BD
service representative.
Possible causes Recommended solutions
Threshold is not set to the
correct parameter (usually
FSC)
Set the threshold to the correct
parameter for your application.
Threshold level is too high Lower the threshold level.
PMT voltage for threshold
parameter is set too low
Set the PMT voltage higher for the
threshold 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 the
flow cell
Prime the fluidics system. See Priming
the fluidics (page 39).
BD LSRFortessa X-20 Cell Analyzer User’s Guide120
No sample in the tube Verify that sample remains in the tube
and if necessary, add sample to the
tube 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 to
display.
Sample injection tube is
clogged
Remove 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 to
warm 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 the
threshold to an alternative laser while
running the appropriate sample. If
unsuccessful, contact BD Biosciences.
Tube is cracked or misshapen Replace the sample tube.
Possible causes Recommended solutions
Chapter 8: Troubleshooting 121
No events in
acquisition display
and RUN button is
orange
Possible causes Recommended solutions
RUN is not activated Press the RUN button.
Sample tube is not installed
or is not properly seated
Install the sample tube correctly on the
SIP.
Waste container is
pressurized
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 by
rotating counter-clockwise.
3. Install a new waste vent filter.
Sample tube is cracked Replace the sample tube.
Waste tubing line is not
connected to the waste cap
Connect the waste tubing line to the
waste cap.
Sheath container is not
pressurized
Ensure that the sheath container lid
and all connectors are securely
seated.
Inspect the sheath container O-ring
inside the lid and replace it if
necessary.
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).
BD LSRFortessa X-20 Cell Analyzer User’s Guide122
No fluorescence
signal
No signal in red
laser channels
High event rate
Possible causes Recommended solutions
Incorrect fluorochrome
assignment
Make sure that the cytometer
configuration in the software matches
the optical filters in the cytometer and
the configuration is as expected.
Wrong filter is installed Make sure the appropriate filter is
installed 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 to
a change in the sheath tank
fluid level
Check the fluid level in the sheath
tank and refill if necessary.
Adjust the laser delay settings. See
Manual settings (page 165).
Possible causes Recommended solutions
Air bubbles in the sheath
filter or flow cell
Remove the air bubbles. See Removing
air bubbles (page 33).
Threshold level is too low Increase the threshold level. See the
BD FACSDiva Software Reference
Manual for instructions.
PMT voltage for the
threshold parameter is set
too high
Set the PMT voltage lower for the
threshold parameter. See the
BD 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.
Chapter 8: Troubleshooting 123
Low event rate
Erratic event rate
Possible causes Recommended solutions
Threshold level is too high Lower the threshold level. See the
BD FACSDiva Software Reference
Manual for instructions.
Air bubble or debris in the
flow cell
Prime the fluidics system. See Priming
the fluidics (page 39).
PMT voltage for the
threshold parameter is set
too low
Set the PMT voltage higher for the
threshold parameter. See the
BD 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 flow
rate setting is not critical to the
application, set the flow rate switch to
MED or HI.
Sample injection tube is
clogged
Remove the sample tube to allow
backflushing.
If the event rate is still erratic, clean
the sample injection tube. See Cleaning
the fluidics (page 47).
Possible causes Recommended solutions
Sample tube is cracked Replace the sample tube.
Air bubble or debris in the
flow 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).
BD LSRFortessa X-20 Cell Analyzer User’s Guide124
Distorted scatter
parameters
Excessive amount
of debris in display
Sample injection tube is
clogged
Remove the sample tube to allow
backflushing.
If the event rate is still erratic, clean
the sample injection tube. See Cleaning
the fluidics (page 47).
Contaminated sample Prepare the specimen again. Ensure
that the tube is clean.
Sheath filter is dirty Replace the filter. See Changing the
sheath filter (page 56).
Possible causes Recommended solutions
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 the
system (page 50).
Air leak at sheath container Ensure that the sheath container lid is
tight 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 the
sheath filter (page 56).
Flow cell is dirty Flush the system. See Flushing the
system (page 50).
Chapter 8: Troubleshooting 125
High CV or poor QC
results
Dead cells or debris in the
sample
Examine the sample under a
microscope.
Sample is contaminated Re-stain the sample. Ensure that the
tube is clean.
Stock sheath fluid is
contaminated
Rinse the sheath container with DI
water, then fill the container with
sheath fluid from another (or new lot)
bulk container.
Possible causes Recommended solutions
Possible causes Recommended solutions
Air bubble in sheath filter or
flow 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 is
tight and all connectors are secure.
Flow cell is dirty Flush the system. See Flushing the
system (page 50).
The dichroic mirrors are
installed backwards
Remove the dichroic mirrors, reverse
them, and replace them in the optical
holder.
BD LSRFortessa X-20 Cell Analyzer User’s Guide126
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 by
rotating counter-clockwise.
3. Install a new waste vent filter.
Poor sample preparation Repeat sample preparation.
Sample was not diluted in the
same 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 the
correct filters.
Old or contaminated QC
particles
Make new QC samples and perform
the quality control procedure again.
Instrument is not warmed up Wait 30 minutes for the instrument to
warm up.
Laser is not functioning Contact BD Biosciences.
Optical alignment problem Contact BD Biosciences.
Optical filters are incorrect Check the configuration and insert the
correct filters.
Possible causes Recommended solutions
Chapter 8: Troubleshooting 127
Electronics troubleshooting
Introduction This topic describes possible problems and recommended solutions
for BD
LSRFortessa X-20 electronic issues.
“Cytometer
Disconnected” in
cytometer window
Possible causes Recommended solutions
Cytometer power is off Turn on the cytometer main power.
Communication failure
between workstation and
cytometer
1. In BD FACSDiva software, select
Cytometer > Connect.
2. If connecting does not work,
restart the cytometer. Turn the
cytometer off, wait 1 minute, and
turn on the cytometer main power.
3. If connecting still does not work,
contact BD Biosciences.
This page intentionally left blank
BD LSRFortessa X-20 Cell Analyzer User’s Guide130
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. An
interactive fluorescence viewer is also available at
bdbiosciences.com.
Designing
multicolor panels
The special order BD LSRFortessa X-20 cell analyzer is designed
specifically for multicolor research. There are many options for
dyes, reagents, and system configurations.
When choosing a configuration or panel, it is important to
remember that final results depend on the excitation and emission
spectra of the individual dye, the number of fluorescently labeled
binding sites on the cell, as well as spectral overlap and spillover to
other 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 131
Example laser and
dye 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)
Red (640 nm)
Yellow-Green
(561 nm)
Blue (488 nm)
Available only on
Special Order
Research Products
BD LSRFortessa X-20 Cell Analyzer User’s Guide132
About configuration maps
Introduction This topic describes the filter and mirror arrangements in the
detector 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 filled
with a blank optic holder, that position on the configuration map is
unlabeled.
Chapter 9: Detector array configurations 133
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 to
provide up to 18-color detection.
Number
of lasers Colors Lasers See section...
1 4 4 Blue Four-color blue laser configuration (page 139)
2 6 4 Blue Four-color blue laser configuration (page 139)
2 Red Two-color red laser configuration (page 142)
75 Blue Five-color blue laser configuration (page 140)
2 NUV Two-color NUV laser configuration (page 148)
85 Blue Five-color blue laser configuration (page 140)
3 Red Three-color red laser configuration (page 143)
BD LSRFortessa X-20 Cell Analyzer User’s Guide134
3 8 4 Blue Four-color blue laser configuration (page 139)
2 Red Two-color red laser configuration (page 142)
2 NUV Two-color NUV laser configuration (page 148)
84 Blue Four-color blue laser configuration (page 139)
2 Red Two-color red laser configuration (page 142)
2 Violet Two-color violet laser configuration (page 145)
10 5 Blue Five-color blue laser configuration (page 140)
3 Red Three-color red laser configuration (page 143)
2 NUV Two-color NUV laser configuration (page 148)
11 5 Blue Five-color blue laser configuration (page 140)
3 Red Three-color red laser configuration (page 143)
3 Violet Three-color violet laser configuration (page 146)
14 5 Blue Five-color blue laser configuration (page 140)
3 Red Three-color red laser configuration (page 143)
6 Violet Six-color violet laser configuration (page 147)
Number
of lasers Colors Lasers See section...
Chapter 9: Detector array configurations 135
4104 Blue Four-color blue laser configuration (page 139)
2 Red Two-color red laser configuration (page 142)
2 Violet Two-color violet laser configuration (page 145)
2 NUV Two-color NUV laser configuration (page 148)
12 2 Blue Two-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)
13 5 Blue Five-color blue laser configuration (page 140)
3 Red Three-color red laser configuration (page 143)
3 Violet Three-color violet laser configuration (page 146)
2 NUV Two-color NUV laser configuration (page 148)
16 5 Blue Five-color blue laser configuration (page 140)
3 Red Three-color red laser configuration (page 143)
6 Violet Six-color violet laser configuration (page 147)
2 NUV Two-color NUV laser configuration (page 148)
18 6 Blue Six-color blue laser configuration (page 141)
4 Red Four-color red laser configuration (page 144)
6 Violet Six-color violet laser configuration (page 147)
2 NUV Two-color NUV laser configuration (page 148)
Number
of lasers Colors Lasers See section...
BD LSRFortessa X-20 Cell Analyzer User’s Guide136
Base configuration The special order BD LSRFortessa X-20 cell analyzer has one base
configuration at installation. Custom configurations can be added
for different applications. The following image shows a default
base cytometer configuration.
Chapter 9: Detector array configurations 137
Upgrade options The special order BD LSRFortessa X-20 cell analyzer optical
configuration can be upgraded to use different special order
wavelength options and a variety of detectors. See
Special order
configurations (page 150) for a list of some common special order
laser/detector combinations available through the BD special order
research program. Many other special order configurations and
options 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 for
standard polygon configurations.
About the maps The tables in this section show the detectors, filters, and mirrors
used in each configuration, and recommended fluorochromes for
each detector. The word “blank” indicates that a blank optical
holder should be used instead of an optic holder containing a
mirror or filter. A dash (—) indicates that no slot exists for a mirror
in that PMT position.
BD LSRFortessa X-20 Cell Analyzer User’s Guide138
Two-color blue
laser configuration
The following map shows the two-color configuration for the
488-nm blue laser.
PMT LP mirror BP filter Fluorochromes
A 640 695/40 PerCP-Cy5.5
B 505 525/50 FITC
C Blank 488/10 SSC
525/50
505LP
695/40
488/10
640LP
Chapter 9: Detector array configurations 139
Four-color blue
laser configuration
The following map shows the four-color configuration for the
488-nm blue laser.
PMT LP mirror BP filter Fluorochromes
A 750 780/60 PE-Cy™7
B 685 695/40 PerCP-Cy5.5, PE-Cy™5, PerCP, PI
C 550 575/26 PE
D 505 530/30 FITC, Alexa Fluor™ 488
E Blank 488/10 SSC
695/40
685LP
530/30
780/60
575/26
488/10
50
5LP
75
0LP
550LP
BD LSRFortessa X-20 Cell Analyzer User’s Guide140
Five-color blue
laser configuration
The following map shows the five-color configuration for the
488-nm blue laser.
PMT LP mirror BP filter Fluorochromes
A 750 780/60 PE-Cy7
B 685 695/40 PerCP-Cy5.5
C 600 610/20 BD Horizon™ PE-CF594,
PE-Texas Red®
D 550 575/26 PE
E 505 530/30 FITC, Alexa Fluor™ 488
F Blank 488/10 SSC
695/40
685LP
575/26
488/10
780/60
610/20
530/30
550
LP
750LP
60
0LP
505LP
Chapter 9: Detector array configurations 141
Six-color blue laser
configuration
The following map shows the six-color configuration for the
488-nm blue laser.
PMT LP mirror BP filter Fluorochromes
A 750 780/60 PE-Cy7
B 685 695/40 PerCP-Cy5.5
C 655 670/30 PE-Cy5
D 600 610/20 PE-CF594, PE-Texas Red®
E 550 575/26 PE
F 505 530/30 FITC, Alexa Fluor™ 488
G Blank 488/10 SSC
695/40
685LP
610/20
530/30
780/60
670/30
575/26
4
88/
1
0
600
LP
505LP
750LP
65
5LP
550 LP
BD LSRFortessa X-20 Cell Analyzer User’s Guide142
Two-color red laser
configuration
The following map shows the two-color configuration for the
640-nm red laser.
PMT LP mirror BP filter Fluorochromes
A 750 780/60 APC-Cy7, APC-H7
B Blank 670/14 APC
670/14
780/60
750LP
Chapter 9: Detector array configurations 143
Three-color red
laser configuration
The following map shows the three-color configuration for the
640-nm red laser.
PMT LP mirror BP filter Fluorochromes
A 750 780/60 APC-Cy7, APC-H7
B 710 730/45 Alexa Fluor™ 700
C — 670/14 APC
730/45
710LP
780/60
670/14
750LP
BD LSRFortessa X-20 Cell Analyzer User’s Guide144
Four-color red laser
configuration
The following map shows the four-color configuration for the
640-nm red laser.
PMT LP mirror BP filter Fluorochromes
A 750 780/60 APC-Cy7, APC-H7
B 710 730/45 Alexa Fluor™ 700
C 675 685/35 Alexa Fluor™ 680
D — 670/14 APC
730/45
710LP
670/14
780/60
685/35
750LP
67
5LP
Chapter 9: Detector array configurations 145
Two-color violet
laser configuration
The following map shows the two-color configuration for the
405-nm violet laser.
PMT LP mirror BP filter Fluorochromes
A 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
525/50
475LP
BD LSRFortessa X-20 Cell Analyzer User’s Guide146
Three-color violet
laser configuration
The following map shows the three-color configuration for the
405-nm violet laser.
PMT LP mirror BP filter Fluorochromes
A 595 610/20 Qdot® 605, BD Horizon™
Brilliant Violet™ BV605
B 475 525/50 AmCyan, V500-C, BV510,
Qdot® 525
C — 450/50 Pacific Blue™, V450, BV421,
Marina Blue®, Alexa Fluor™ 405
525/50
475LP
610/20
450/50
595LP
Chapter 9: Detector array configurations 147
Six-color violet
laser configuration
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® 655
B 595 610/20 BV605, Qdot® 605
C 575 585/15 Qdot® 585
D 545 560/20 Qdot® 565
E 475 525/50 AmCyan, V500-C, BV510, Qdot® 525
F Blank 450/50 Pacific Blue™, V450, BV421,
Marina Blue®, Alexa Fluor™ 405
610/20
595LP
560/20
450/50
655/8
585/15
525/50
54
5LP
63
0LP
575LP
475LP
BD LSRFortessa X-20 Cell Analyzer User’s Guide148
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 Fluorochromes
A 505 530/30 Indo-1 (Blue)
B Blank 450/50 Indo-1 (Violet), DAPI,
Alexa Fluor™ 350
450/50
530/30
505LP
Chapter 9: Detector array configurations 149
Five-color yellow-
green laser
configuration
The following map shows the five-color configuration for the
561-nm yellow-green laser.
More information • About the base configuration (page 133)
• Special order configurations (page 150)
PMT LP mirror BP filter Fluorochromes
A 750 780/60 PE-Cy7
B 685 710/50 PerCP-Cy5.5
C 635 670/30 PE-Cy5
D 600 610/20 PE-CF594, PE-Texas Red®
E Blank 586/15 PE
710/50
685LP
610/20
780/60
670/30
586/15
600LP
75
0LP
635LP
BD LSRFortessa X-20 Cell Analyzer User’s Guide150
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
CS&T
Not all combinations of special order BP filters and lasers are
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 with
this laser wavelength for cell surface markers, live/dead
discrimination, 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™ 488 530/30
PE 575/25
575/26
576/26
585/42
Chapter 9: Detector array configurations 151
Red 640 nm
(40
mW)
The fluorochromes listed in the following table can be used with
this laser wavelength for the cell surface marker application. See
Common red laser configurations (page 157) for two common
polygon configuration maps.
PE-CF594, PE-Texas Red®, PI 605/12
610/20
616/23
PE-Cy5 660/20
670/30
PerCP, PerCP-Cy5.5, PE-Cy5.5 670/14
675/20
695/40
710/50
PE-Cy7 780/60
Fluorochromes BP filters
Fluorochromes BP filters
APC, Alexa Fluor™ 647 670/30
Alexa Fluor™ 680 685/35
Alexa Fluor™ 700 710/50
710/20
712/21
730/45
APC-Cy7, APC-H7 780/60
BD LSRFortessa X-20 Cell Analyzer User’s Guide152
Violet 405 nm
(20–100 mW)
The fluorochromes listed in the following table can be used with
this laser wavelength for cell surface marker, live/dead
discrimination, and cell cycle applications. See
Common violet
laser configurations (page 158) for common polygon configuration
maps.
Note: 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.
Fluorochromes BP filters
V450, BV421, Pacific Blue™ 450/50
450/40
450/20
440/40
AmCyan, Alexa Fluor™ 430, V500-C,
BV510, DAPI
510/50
525/50
Qdot® 525 NA
Qdot® 545 NA
Qdot® 565 560/20
560/40
Qdot® 585 585/15
585/42
BV605, Qdot® 605 605/12
605/40
610/20
Qdot® 655 655/8
670/30
BD Horizon™ Brilliant Violet™ BV711,
Qdot® 700, Qdot® 705
710/50
712/20
Qdot® 800 780/60
800/30
Chapter 9: Detector array configurations 153
UV 355 nm (20–
100
mW)
The fluorochromes listed in the following table can be used with
this 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.
Yellow-green 561
nm (50
mW)
The fluorochromes listed in the following table can be used with
this laser wavelength for cell surface marker and fluorescent
protein applications. See
Common yellow-green laser
configuration (page 162) for a common polygon configuration
map.
Fluorochromes BP filters
DAPI, Hoechst 440/40
450/20
450/40
450/50
Indo-1 (Ca Bound) 450/50
Indo-1 (Ca unbound) 530/30
Fluorochromes BP filters
PE, DsRed 586/15
PE-CF594, PE-Texas Red®,
mCherry
610/20
PE-Cy5 660/20
670/30
PE-Cy5.5 710/50
PE-Cy7 780/60
BD LSRFortessa X-20 Cell Analyzer User’s Guide154
Green 532 nm
(150
mW)
The fluorochromes listed in the following table can be used with
this laser wavelength for the cell surface markers application. See
Common green laser configuration (page 163) for a common
polygon configuration map.
Fluorochromes BP filters
PE 575/25
PE-CF594, PE-Texas Red® 610/20
PE-Cy5 660/20
670/30
PE-Cy5.5 695/40
710/50
PE-Cy7 780/60
Chapter 9: Detector array configurations 155
Common blue laser
configurations
The following maps show two common configurations for the
488-nm blue laser.
710/50
685LP
575/26
488/10
780/60
610/20
525/50
55
0LP
73
5LP
600LP
505 LP
FITC
SSC
PE
PerCP-Cy5.5
PE-CF594 PE-Cy7
BD LSRFortessa X-20 Cell Analyzer User’s Guide156
Note: With the 488-nm blue laser, FITC requires a 525/50 notch
filter if the cytometer also uses a 532-nm laser.
525/50
505LP
710/50
488/10
68
5LP
FITC
SSC PerCP-Cy5.5
Chapter 9: Detector array configurations 157
Common red laser
configurations
The following maps show two common configurations for the
640-nm red laser.
670/30
780/60
735LP
APC
APC-Cy7
730/45
685LP
780/60
670/30
735LP
Alexa Fluor
™
700
APC-Cy7
APC
BD LSRFortessa X-20 Cell Analyzer User’s Guide158
Common violet
laser
configurations
The following maps show two common configurations for the
405-nm violet laser.
450/50
525/50
505LP
V450 or BV421
AmCyan or BV510
Chapter 9: Detector array configurations 159
Note: If a 561-nm yellow-green laser is configured, a 561 notch
filter is required for Qdot® 565 and Qdot® 585.
710/50
685LP
610/20
560/40
450/50
780/60
670/30
585/42
525/50
60
0LP
545LP
73
5LP
635LP
570 LP
505LP
Qdot
®
585
V500-C or BV510
Qdot
®
565
V450 or
BV421
BV605
BV711
Qdot
®
655 Qdot
®
800
BD LSRFortessa X-20 Cell Analyzer User’s Guide160
Common UV laser
configurations
The following maps show two common configurations for the
355-nm UV laser.
450/50
525/50
50
5LP
Indo-1 (violet)
Indo-1 (blue)
Chapter 9: Detector array configurations 161
525/50
505LP
670/50
450/50
630LP
Indo-1 (blue)
Hoechst RedIndo-1 (violet)
BD LSRFortessa X-20 Cell Analyzer User’s Guide162
Common yellow-
green laser
configuration
The following map shows a common configuration for the 561-nm
yellow-green laser.
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.
710/50
685LP
610/20
780/60
660/20
586/15
60
0LP
73
5LP
630LP
570LP
PE
PE-CF594
PE-Cy5.5
PE-Cy5 PE-Cy7
Chapter 9: Detector array configurations 163
Common green
laser configuration
The following map shows a common configuration for the 532-nm
green laser.
710/50 or
695/40
685LP
610/20
780/60
660/20
585/42
60
0LP
73
5LP
630LP
PE
PE-CF594
PE-Cy5.5
PE-Cy5 PE-Cy7
This page intentionally left blank
BD LSRFortessa X-20 Cell Analyzer User’s Guide166
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. Laser
light 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 cross-
contamination 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 red
lasers. Because the laser signals are spatially separated, there is a
slight delay between the detection of each laser’s signal.
The laser delay setting in BD FACSDiva software is used to re-align
the signals so they can be measured and displayed on the same time
scale. 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 the
longest delay.
Time
Red
Blue
UV
Violet
Event intercepts blue laser
Event intercepts violet laser
Event intercepts UV laser
Event intercepts red laser
Event intercepts yellow-green laser*
Yellow-green
*The yellow-green laser is only available through the BD special order research program.
Chapter 10: Manual settings 167
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 events
from 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 to
show the fluorescence signal excited by the laser for which the
delay 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).
BD LSRFortessa X-20 Cell Analyzer User’s Guide168
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 fluorescence
intensity (MFI) of the gated population.
9. While observing the MFI for the gated population, adjust the
laser delay in 0.1-µs increments within a range of 2.0
µs of the
setting obtained in
step 6.
Preserve the setting that maximizes the fluorescence intensity.
10. Reset the Window Extension to 10 µs.
Chapter 10: Manual settings 169
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 to
verify that the area calculation and the height measurement are
equivalent 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 your
experiment. To adjust the other lasers, add a parameter and the
corresponding 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 button
on the Browser toolbar.
BD LSRFortessa X-20 Cell Analyzer User’s Guide170
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 and
histograms:
• 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 171
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 to
tube_001.
BD LSRFortessa X-20 Cell Analyzer User’s Guide172
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 on
scale.
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 is
lower than FSC-H.
Chapter 10: Manual settings 173
• Decrease the area scaling factor if the FSC-A signal is
higher than FSC-H.
c. View the result of your change in the histograms and
statistics 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 APC-
positive control tube.
BD LSRFortessa X-20 Cell Analyzer User’s Guide174
14. Adjust the red laser area scaling factor until the APC-A signal
matches the APC-H signal, if needed.
BD LSRFortessa X-20 Cell Analyzer User’s Guide176
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 559123
SPHERO 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 177
CS&T beads
Reagents
Bead Laser Supplier Catalog No.
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)
BD Biosciences
655050
(50 tests)
655051
(150 tests)
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 660585
BD LSRFortessa X-20 Cell Analyzer User’s Guide178
Equipment
BD FACSClean solution BD Biosciences 340345
Dyes and fluorochromes BD Biosciences, Life
Technologies, or Sigma
–
Chlorine bleach
(5% sodium hypochlorite)
Clorox® or other major
supplier (to ensure that the
bleach is at the correct
concentration and free of
particulate matter)
–
Reagent Supplier Catalog No.
Equipment item Supplier Catalog No.
Bal seal BD Biosciences 343509
O-ring, sample tube 343615
Sheath filter assembly 345734
Falcon polystyrene test tubes, 12 x 75-mm Corning 352008
Index
Numerics
355-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, 155
532-nm green laser 154, 163
561-nm yellow-green laser 153, 162
640-nm red laser 142–144, 151, 157
A
acridine orange (AO) 47
air bubbles, removing 33
alarm
battery test 52
waste container 23
analysis
data 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 13
B
Bal seal
ordering 178
replacing 58
bandpass (BP) filters
detector array placement 25
theory 107
base configurations 137
battery
changing 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
C
calculating compensation 82
capacity, waste container 23
BD LSRFortessa X-20 Cell Analyzer User’s Guide180
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
configuration
base 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 22
E
electronics
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
Index 181
specifying parameters 73
F
FACSFlow sheath fluid 177
FACSFlow solution 33
Falcon tubes 119
filters, optical
bandpass (BP) 25, 107
changing 42
dichroic See dichroic mirrors
longpass (LP) 104, 105
notch 105, 156
shortpass (SP) 104, 106
theory 104
filters, sheath
attaching new 57
replacing 56
FITC and Stokes shift 103
fixed-alignment lasers 16
flow cell
draining 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
fluorochromes
compensation theory 109
emission spectra 103
measured 41
role in light emission 102
flushing the system 50
forward scatter (FSC) 102
FSC See forward scatter
G
gating data 91
global worksheets
analyzing data 91
creating 87
previewing data 86, 97
green 532-nm laser 154, 163
H
hazard symbol definitions 11
Help, accessing 12
High Throughput Sampler (HTS) 22
hydrodynamic focusing 100
I
immunophenotyping
analysis 91
experiment 87
hydrodynamic focusing 101
K
knob, SAMPLE FINE ADJ 19
L
laser delay
about 166
optimizing 167
lasers
BD LSRFortessa X-20 Cell Analyzer User’s Guide182
options 24
performance check 68
quality control (QC) particles 176
longpass (LP) filters
defined 104
theory 105
LP See longpass filters
M
maintenance
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 25
N
notch filters 105, 156
O
online Help 12
optic holder 41
optics
components 23
configuration 66, 67
dichroic mirrors 108
filters 25, 42, 104
location 17
optimization, sample 64
ordering spare parts 176
O-ring
ordering 178
replacing 60
worn 118
P
particles 176, 177
PerCP, Stokes shift 102
performance check 68, 69
photodiode 26
photomultiplier tubes (PMTs) 26
PMT See photomultiplier tubes
polygon
location 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
Q
quality control (QC)
particles 176
troubleshooting 125
R
recording
compensation 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
Index 183
sheath 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
sample
optimization 64
optimization experiment 72
optimization, single-stained
controls 65, 86
SAMPLE FINE ADJ knob 19
sample injection port (SIP)
components 21
hydrodynamic 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
scatter
light 102
parameter distortion 124
setup, cytometer 64
sheath
flow 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
software
cytometer 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
T
technical 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
U
user preferences 67
BD LSRFortessa X-20 Cell Analyzer User’s Guide184
UV 355-nm laser 153, 160
V
violet 405-nm laser 145–147, 152, 158
W
waste 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
Y
yellow-green 561-nm laser 153, 162
