BD FACSCelesta™ Flow Cytometer User's Guide

BD FACSCelesta™ Flow
Cytometer
User’s Guide
For Research Use Only
23-17147-02
9/2020
Becton, Dickinson and Company BD Biosciences

BD Biosciences European Customer Support
2350 Qume Drive Tel +32.53.720.600
San Jose, CA 95131 USA [email protected]
bdbiosciences.com
[email protected]
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
Clorox is a registered trademark of The Clorox Company.
SPHERO is a trademark of Spherotech, Inc.
Microsoft and Windows are registered trademarks of Microsoft Corporation.
Texas Red® and Alexa Fluor® are registered trademarks of Life Technologies Corporation.
Teflon is a registered trademark of E.I. du Pont de Nemours and Company.
Living Colors® (DsRed, mCherry) is a registered trademark of Clontech.
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.
Falcon® is a registered trademark of Corning Incorporated.
CF™ is a trademark of Biotium, Inc.
BD, the BD Logo, FACS, FACSCelesta, FACSClean, FACSDiva and FACSFlow 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.
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
23-17147-00 11/2015 Initial release
23-17147-01 6/2016 Updated the guide by removing status screen image and air
pressure definition.
23-17147-02 9/2020 Removed references to 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 FACSCelesta documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Instrument technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Chapter 2: Introduction 15
System overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Cytometer overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Control panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Fluidics system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sheath and waste containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Chapter 3: Cytometer setup 31

Starting the cytometer and computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Preparing the sheath container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Removing air bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Preparing the waste container . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Priming the fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
About the optical filters and mirrors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Custom configurations and baselines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
vi BD FACSCelesta Flow Cytometer User’s Guide
Chapter 4: Maintenance 45
Maintenance overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Cleaning the fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Shutting down the cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Flushing the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Replacing the waste container cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Changing the sheath filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Changing the Bal seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Changing the sample tube O-ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Cleaning or replacing the sheath gasket . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Chapter 5: Optimizing cytometer settings 61

Cytometer settings workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Verifying the configuration and user preferences . . . . . . . . . . . . . . . . . . . . . . . . . 64
Running a performance check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Setting up an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Creating application settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Recording compensation controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Calculating compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
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 vii
Chapter 8: Troubleshooting 117

Cytometer troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Electronics troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Chapter 9: Detector array configurations 127

Fluorescence spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
About configuration maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
About the configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Base configuration polygon maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Chapter 10: Supplies and consumables 143

Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Index 147
1
About this guide
This chapter covers the following topics:
• What this guide covers (page 10)

• Conventions (page 11)
• About the BD FACSCelesta documentation (page 11)
• Instrument technical support (page 13)
10 BD FACSCelesta Flow Cytometer User’s Guide
What this guide covers

This guide describes the procedures necessary to operate and
maintain your BD FACSCelesta™ flow cytometer. 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.
Conventions
Introduction The following table lists the safety symbols used in this guide to
alert you to potential hazards.
Safety symbols
Symbol Meaning
Caution! 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
About the BD FACSCelesta documentation

Introduction This topic describes the documentation available with the
BD FACSCelesta flow cytometer.
Publication formats This guide is provided in PDF format to provide an eco-friendly

option. All content is also included in the BD FACSDiva software
Help.
Help system The help system installed with BD FACSDiva software includes all
content from this guide and the documents listed below. Access the
BD FACSCelesta help system from the Help menu in the
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 FACSCelesta Flow Cytometer Site Preparation Guide:
Contains specifications for:
– Cytometer weight and size
– Temperature
– 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.
Additional BD Cytometer Setup and Tracking Application Guide: Describes

Documentation how to use the BD™ Cytometer Setup and Tracking (CS&T)
features in BD FACSDiva software. You can find the document in
the following location bdbiosciences.com/guide.
Instrument technical support

Introduction This topic describes how to get technical assistance.
Contacting If technical assistance is required, contact your local

technical support 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 will see information in your local

language.
3. Click Go.
4. Click the Support link for details for your local region.
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2
Introduction
• System overview (page 16)

• Control panel (page 19)
• Fluidics system (page 20)
• Sheath and waste containers (page 27)
• Optics (page 28)
• Workstation (page 30)
System overview
About the system The BD FACSCelesta system includes the BD FACSCelesta flow
cytometer, BD FACSDiva software v8.0.1.1 running on the system
workstation, the optional BD FACSFlow™ supply system (FFSS),
and the optional BD High Throughput Sampler (HTS). Each
component is described in detail in the following sections.
1 2 3
Number Components
1 Sheath and waste tanks
2 BD FACSCelesta flow cytometer
3 Computer workstation
Cytometer overview
Introduction The BD FACSCelesta flow cytometer is an air-cooled multi-laser
benchtop flow cytometer with the ability to acquire 10–12
fluorescent parameters. It uses fixed-alignment lasers that transmit
light through a flow cell to collect and translate the resulting
fluorescence signals into electronic signals. Cytometer electronics
convert these signals into digital data.
Components The following figure shows the main components of the

BD FACSCelesta flow cytometer, which are listed in the table. Each
component is described in detail in the following sections.
2
3
7 8
Number Component
1 Heat ventilation slots (top)
2 Control panel
3 Power button
4 Electrical plug
5 Fluidic sensor ports
6 Sample injection port (SIP)
7 Heat ventilation slots (left side)
8 Air and fluidic ports
9 Optics access door (polygon

detector arrays)
Caution! Do not place any objects on top of the

instrument. Blocking the ventilation may cause the
instrument to overheat.
Caution: Electrical Hazard! Do not place liquids on top of

the instrument. Any spill of liquid into the ventilation
openings could cause electrical shock or damage to the
instrument.
Control panel
Overview The following figure shows the components in the control panel,
which are listed in the table.
1 2 5
3 4 6
Number Component
1 System indicators
2 Fluid control buttons
3 Sample flow rate buttons

Number Component
4 Sample fine adjust buttons
5 Status screen
6 MODE button
More information • Fluidics system (page 20)

• Optics (page 28)
Fluidics system
Introduction The fluidics system carries the sample out of the sample tube and
into the interrogation region of the flow cell. Cells are carried in
the sample core stream in single file and measured individually.
System indicators There are two system indicators (System Status and Activity) on
the control panel.
• System Status. Shows the status of the sheath and waste tank
levels. The following table describes the LED indicators,
conditions that trigger them, and any action that must be
taken.
LED color Status Action Condition

Green Good None Good
Yellow Caution! Sheath Check tank levels Sheath low and/or

and/or waste tanks waste nearly full
need attention.
Red Caution! Take  Empty waste tank Sheath empty and/or

immediate action.  Fill sheath tank waste full
System status is also displayed on the Status screen. See Status

screen (page 23) for a description of the Status screen.
• Activity. Shows whether the cytometer power is on and the

status of acquisition. The following table describes the
indicator LEDs, and status that triggers them.
Indicator LED color Status

Steady pulse blue Cytometer is powered on
Fluctuates blue Cells are passing through the

flow cell
Fluid control The three fluid control buttons (RUN, STANDBY, and PRIME) set
the cytometer operation.
• 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.
The RUN button will also turn orange if the sample tube is
cracked or the BAL seal is bad. See Troubleshooting
(page 117).
• STANDBY. Stops fluid flow to conserve sheath fluid.
When you leave the cytometer for more than a few minutes,
place a tube containing less than 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.
Note: Remove sample tubes during the prime cycle.
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.
Sample flow rate The three flow rate control buttons (LOW, MED, HIGH) set the
control sample flow rate through the flow cell. The SAMPLE ADJ buttons
allow you to adjust the rate to intermediate levels.
2 3
Number Component
1 Status screen
2 Sample flow rate buttons
3 Sample fine adjust buttons
When sample adjust is set to 250 (as shown on the status screen on
the control panel) the sample flow rates at the Low, Med, and High
settings are approximately 12, 35, and 60 µL/min of sample,
respectively. Each time you press one of the SAMPLE ADJ buttons,
the fine adjust of the indicated sample increases or decreases by 10.
The following table shows the approximate sample flow rate range
for low, medium, and high.
Settings Sample flow rate (µL/min)

Low 6–24
Med 17.5–70
High 30–120
Status screen The status screen line toggles between two different displays and is
described in detail in the following table.
2 1
4
Line Definition
1 Waste level. Shows range from E (empty) to F (full). The
display line increases from left to right in sequences of
20%. System status turns yellow at 80%, and red at 100%
full.
2 Sheath level. Shows range from E to F. The display line

decreases from right to left in sequences of 20% from full
level. System status turns yellow at 20%, and red at 0%.
3 Fine Adj. Shows the current setting of fine adjust. Fine

adjust can be set in increments of 10 from 0 to 500. The
normal set point is 250. The last value persists, even after
cytometer shutdown.
4 HTS mode. Shows that the cytometer is in HTS mode. To

enter this mode, press and hold down the MODE button
for more than 3 seconds. Status information is not
displayed and system status turns green. To return to the
normal mode, press and hold the MODE button for more
than 3 seconds
Fluidic alarms and The fluidic alarms are triggered by the waste and sheath fluid levels
the Mode button in the tanks. The alarms sound when the waste tank is nearly
100% full and the sheath tank is empty. The fluidic alarms and
system status will also show warnings when you start up the
cytometer until the tank pressure reaches the correct level.
To silence the alarm, press the MODE button, then press the Down
button. The MODE button flashes to indicate the cytometer is in
silent mode. Repeat this sequence to turn off silent mode.
Note: Turning the cytometer to silent mode may result in overflow

of waste and you may run out of sheath.
Note: When the cytometer is in HTS mode, both visual and

audible alarms will be deactivated.
Sample injection The SIP is where the sample tube is installed. The SIP includes the
port (SIP) 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.
Outer sleeve
Sample injection tube
Tube support arm
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.
Cautions when
using the HTS Caution: Biohazard! When using the BD FACSCelesta
option flow cytometer 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.
Droplet The DCM prevents sheath fluid from dripping from the SIP and
containment provides biohazard protection.
module
The DCM vacuum is activated when the tube support arm is
moved to the side. Sheath fluid is aspirated as it backflushes the
sample injection tube. This backflush helps prevent carryover of
cells between samples.
Sheath and waste containers

Introduction This topic describes the sheath and waste containers. The sheath
and waste containers are outside the cytometer and can be
positioned on the floor.
Note: Only BD service engineers should change the location of the

sheath and waste tanks.
If you are using the BD FACSFlow™ supply system (FFSS), see the
documentation provided.
Sheath container The sheath container has a capacity of 10 L. Sheath fluid is filtered
through an in-line, interchangeable filter that prevents small
particles from entering the sheath fluid lines. An alarm sounds
when the container is empty.
Caution! Do not fill the sheath tank to its maximum

capacity. When an overfull tank is pressurized, erratic
cytometer performance can result.
Waste container The waste container has a capacity of 10 L. An alarm sounds when
the container is full.
More information • Preparing the sheath container (page 33)

• Preparing the waste container (page 38)
• Status screen (page 23)
• Fluidic alarms and the Mode button (page 24)
Optics
Introduction This topic describes the optical components for the
BD FACSCelesta flow cytometer including:
• Detector arrays
• Laser options
• Optical filters
• Signal detectors
Detector arrays The BD FACSCelesta detector arrays consist of polygons. Each

polygon can be outfitted with two to six fluorescent detectors and
can detect up to six fluorescent signals.
Laser options The BD FACSCelesta flow cytometer can be configured with up to

three lasers as listed in the following table.
Laser Wavelength (nm) Power (mW)

Violet (standard) 405 50
Blue (standard) 488 20
UV (optional) 355 15
Yellow Green 561 50

(optional)
Red (optional) 640 40
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 FACSCelesta:
• Longpass dichroic filters (LPs). Transmit wavelengths at or

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. Longpass dichroic mirrors transmit longer wavelengths to
one detector while reflecting shorter wavelengths to a different
detector.
The BD FACSCelesta flow cytometer polygon detector arrays use

dichroic longpass mirrors on the inside, and bandpass filters on the
outside of the filter holders.
Bandpass filters
Longpass dichroic mirrors
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 FACSCelesta

flow cytometer:
• 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.
More information • Optical filter theory (page 104)

• About the configuration (page 132)
Workstation
Introduction This topic describes the components of the BD FACSCelesta
workstation.
Workstation Acquisition, analysis, and most instrument functions are controlled

components by the BD FACSCelesta workstation. It includes a PC and one or
two monitors.
Your workstation is equipped with the following:
• Microsoft Windows operating system

• BD FACSDiva software version 8.0.1.1 or later for data
acquisition and analysis
• Software documentation including the help system
More information • About the BD FACSCelesta documentation (page 11)

3
Cytometer setup
• Starting the cytometer and computer (page 32)

• Preparing the sheath container (page 33)
• Removing air bubbles (page 35)
• Preparing the waste container (page 38)
• Priming the fluidics (page 40)
• About the optical filters and mirrors (page 41)
• Custom configurations and baselines (page 43)
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.
Allow 30 minutes for the optical system temperature to

stabilize.
Caution! Failure to warm up and stabilize the

instrument could affect sample data.
2. 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.
3. Start BD FACSDiva software by double-clicking the shortcut

on the desktop, and log in to the software.
4. 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.
If the message Cytometer Disconnected appears, see

Electronics troubleshooting (page 126).
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, see

the documentation provided with your system.
When to check the Check the fluid levels in the sheath container every time you use
sheath container the cytometer. This ensures that you do not run out of sheath fluid
during an experiment.
Sheath container
components
Air line (green)
Alarm Sensor
Clamp Knob
Cap handle
Vent Valve
Filter assembly
Sheath fluid
line (blue) to
cytometer
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 and blue sheath fluid line from
the sheath container.
3. Disconnect the alarm line from the alarm sensor socket by

pulling lightly on both ends of the plug.
4. Depressurize the sheath container by pulling up on the vent

valve.
5. 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.
6. Add up to 10 L of sheath fluid, such as BD FACSFlow

solution, to the sheath container.
Note: 10 L will reach the interior line on the sheath tank. Do

not fill the sheath tank further.
7. Replace the sheath container lid.
8. Make sure the gasket on the inside lip of the sheath lid is
seated correctly and has not slipped out of position.
Note: 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.
10. Reconnect the green air line, alarm line and the blue sheath
line.
Note: Ensure that the blue sheath fluid line is not kinked.
More information • Removing air bubbles (page 35)

• Changing the sheath filter (page 54)
• Cleaning or replacing the sheath gasket (page 60)
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.
Note: Perform this activity every time the sheath tank is refilled.
Procedure To remove air bubbles:

1. Check the sheath filter for trapped air bubbles.
Cytometer fluid line

(roller clamp not visible)
Vent fitting
Vent line
2. If bubbles are visible, gently tap the filter body with 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.
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.
Button
Vent fitting
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.
Note: The roller clamp can be found close to the fluidics ports
of the cytometer.
8. Close the roller clamp.

Preparing the waste container

Introduction This topic describes how to prepare the waste container. Prevent
waste overflow by emptying the waste container daily or whenever
the system status indicator turns yellow.
Note: If your system is connected to the FFSS, see the

documentation provided with your FFSS.
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.
Waste container The following figure shows the main components of the waste
components container.
User replaceable cap
Alarm sensor
Moisture trap Air line
Waste fluid
line
Biological
precautions Caution: Biohazard! Contact with biological specimens
and materials can transmit potentially fatal disease.
To prevent exposure to biohazardous agents:
• Put the cytometer in standby mode before disconnecting the

waste tank to avoid leakage of biohazardous waste.
• Always disconnect the waste container from the cytometer
before you empty it. Wait at least 30 seconds for pressure to
dissipate before you remove the waste cap or sensor.
• Expose waste container contents to bleach (10% of total
volume) for 30 minutes before disposal.
• Do not wet the waste tank cap. If wet, the filter in the cap will
cause the tank to malfunction. To keep the cap dry, place it on
the bench label side up when it is not on the tank.
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 from the waste container.
3. Disconnect the black alarm sensor line from the alarm sensor
socket.
Keep the lid and moisture trap on the waste container until
you are ready to empty it.
4. Remove the lid and moisture trap before emptying the waste
container. Keep the cap label side up at all times. Empty the
waste container.
Caution! The waste container is heavy when full.

When emptying it, use good body mechanics to
prevent injury.
5. Add approximately 1 L of bleach to the waste container and
close it. Reattach the moisture trap and lid.
6. Reconnect the orange waste tubing and make sure it is not

kinked.
7. Reconnect the black alarm sensor line.
Priming the fluidics

Introduction This topic describes how to prime the fluidics system.
When to prime the Sometimes, air bubbles and debris may become lodged in the flow
fluidics 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 less than 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)

About the optical filters and mirrors

Introduction This topic provides a description of the optical filters and mirrors.
Filter and mirror Each detector has an optic holder in front of it. The optic holders
configurations are labeled with numbers indicating the wavelengths of the
bandpass filter and longpass dichroic mirror they contain (for
example, 780/60 and 750 LP, respectively). The optic holder in
front of the last detector in the detector array contains only a
bandpass filter and is marked accordingly.
PMT A
Optic holder
handle
Optic holders
3
The filters steer progressively shorter wavelengths of light to the

next detector in the array as indicated by the lines and arrows on
the top of the polygon.
Optic holders, Optic holders house filters and mirrors. Your cytometer includes
filters, and mirrors several blank (empty) optic holders.
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.
Caution! Do not remove or change filters. Only BD

authorized personnel should remove or change the filters.
Base configurations Each BD FACSCelesta flow cytometer 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 Before you acquire data using BD FACSDiva software, you must
cytometer specify a cytometer configuration. The cytometer configuration
configuration defines which filters and mirrors are installed at each detector.
BD FACSDiva software provides a BD base configuration for your

BD FACSCelesta flow cytometer. 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 • About the configuration (page 132)

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.
See Optimizing cytometer settings (page 61). 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 67)

4
Maintenance
• Maintenance overview (page 46)

• Cleaning the fluidics (page 47)
• Shutting down the cytometer (page 49)
• Flushing the system (page 50)
• Replacing the waste container cap (page 52)
• Changing the sheath filter (page 54)
• Changing the Bal seal (page 56)
• Changing the sample tube O-ring (page 58)
• Cleaning or replacing the sheath gasket (page 60)
Maintenance overview
Introduction This topic provides an overview of the BD FACSCelesta flow
cytometer routine maintenance and cleaning procedures.
General use
guidelines Caution: Biohazard! Contact with biological specimens
Follow these guidelines whenever operating or maintaining the

cytometer:
• Wear suitable protective clothing, eyewear, and gloves.

• Handle all biological specimens and materials in accordance
with applicable regulations and manufacturer specifications.
• Dispose of waste using proper precautions and in accordance
with local regulations.
• Never pipette by mouth.
For fluidics maintenance, we recommend the following cleaning
solutions:
• BD™ FACSClean solution

• 1.5% BD Detergent Solution Concentrate
• 10% bleach solution
Use DI water to dilute bleach to appropriate concentrations.
Caution! Higher concentrations of bleach and use of

other cleaning solutions might damage the cytometer.
When to perform Perform maintenance procedures in the following frequencies.

maintenance
procedures
Frequency Maintenance procedure
Daily  Cleaning the fluidics (page 47)
 Shutting down the cytometer (page 49)
Scheduled (every  Flushing the system (page 50)

two weeks)
Periodic (frequency  Changing the sheath filter (page 54)

depends on how  Changing the Bal seal (page 56)
often you run the
cytometer)  Changing the sample tube O-ring (page 58)
 Cleaning or replacing the sheath gasket
(page 60)
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 10% bleach solution on the

SIP with the support arm to the side (vacuum on) and let it run
for 1 minute.
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 1.5% BD Detergent Solution

Concentrate.
Note: The BD Detergent Solution Concentrate must be diluted

before use. 1.5% BD Detergent Solution is made from BD
Detergent Solution Concentrate, 15 mL diluted to make 1 L
total.
Caution: Biohazard! Never mix BD Detergent Solution

and bleach because they can create dangerous fumes.
5. Repeat steps 2 and 3 with DI water.
6. Press the STANDBY button on the fluidics control panel.
7. Place a tube containing less than 1 mL of DI water on the SIP.
A tube with less than 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 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.
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. Close FACSDiva software.
4. Select Start > Shutdown to turn off the computer (if needed).
5. If your system is connected to the FFSS, shut off the FFSS.
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)
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
Caution: Biohazard! The cytometer hardware might be
contaminated with biohazardous material. Use 10%
bleach to decontaminate the instrument.
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.
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.
2. Empty the sheath container and rinse it with DI water.
3. Fill the sheath container with at least 1 L of 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.
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 light is amber, press the
PRIME button again.
8. Install a tube with 3 mL of 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.

Ensure that the sample fine adjust is set to 250. 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.
12. Repeat steps 2 through 11 with 1.5% BD Detergent Solution

Concentrate.
Caution: Biohazard! Never mix BD Detergent Solution

and bleach because they can create dangerous fumes.
13. Repeat steps 2 through 11 with DI water.
14. Replace the sheath filter and refill the sheath container with
sheath fluid.
15. Install a tube with less than 1mL DI water onto the SIP.
Replacing the waste container cap

Introduction This topic describes how to replace the waste container cap.
Replace the cap once a month.
Biological
precautions Caution: Biohazard! Contact with biological specimens
To prevent exposure to biohazardous agents:
• Put the cytometer in standby mode before disconnecting the

waste tank to avoid leakage of biohazardous waste.
• The waste container can become pressurized when the
cytometer is running. Always disconnect the waste container
from the cytometer before you empty it. Wait at least
30 seconds for pressure to dissipate before you remove the
waste cap or sensor.
• Expose waste container contents to bleach (10% of total
volume) for 30 minutes before disposal.
Procedure To replace the cap:
1. Put the cytometer in standby mode.
2. Disconnect the orange waste line from the waste container

tank.
3. Disconnect the alarm sensor line from the alarm sensor socket.
Note: Wait at least 30 seconds for pressure to dissipate.
4. Remove the waste cap and attached trap from the container
and place on the bench label-side up.
5. Detach the cap from the trap.
6. Place a new cap on the trap.

7. Write the date on the cap label.
Waste (A)
Space for dat e

338677 Rev A
8. Screw the cap assembly onto the waste container and hand-
tighten until it is fully closed.
Caution: Biohazard! To prevent waste container

overpressurization, do not overtighten the trap or
attached filter cap. Tighten each component only until it
is hand-tight. Do not use sealants such as Teflon® tape
or other adhesives.
Re-attach the alarm sensor line and waste line to the waste
container tank.
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 We recommend changing the sheath filter assembly every

the sheath filter 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 143) for ordering information.
Sheath filter
components
Sheath line
Vent fitting
Quick-disconnect
Vent line
Filter base
Removing the old To remove the old filter:

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 To attach the new filter:

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.

Changing the Bal seal

Introduction This topic describes how to replace the Bal seal.
The sample injection tube Bal seal is a ring thaft forms a seal with
the sample tube and ensures proper tube pressurization.
When to change Over time, the Bal seal becomes worn or cracked and requires
the Bal seal 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).
Caution
Caution: Biohazard! Cytometer hardware might be
contaminated with biohazardous material. Wear suitable
protective clothing, eyewear, and gloves whenever
cleaning the cytometer or replacing parts.
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.
Bal seal
Retainer
Outer sleeve
Sample injection tube
Work carefully. The outer sleeve can fall off as you loosen the
retainer.
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.
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 Replace the O-ring when droplets form at the end of the sample
the O-ring injection tube while the vacuum is operating.
Caution
Caution: Biohazard! Cytometer hardware might be
contaminated with biohazardous material. Wear suitable
protective clothing, eyewear, and gloves whenever
cleaning the cytometer or replacing parts.
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.
O-ring
Retainer
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.
Cleaning or replacing the sheath gasket

Introduction This topic describes how to clean or replace the gasket of the
sheath tank lid.
When to change We recommend cleaning the sheath gasket when needed.

the sheath gasket
Procedure To clean or replace the gasket:

1. Put the cytometer in standby mode.
2. Depressurize the sheath container by pulling up on the vent

valve.
3. Remove the lid from the sheath tank.
4. Remove the black gasket from the lid.
5. Rinse it with water to clean the gasket.
6. Place the clean gasket or the new gasket on the lid and make
sure the gasket is seated properly on the lid.
5
Optimizing cytometer settings
• Cytometer settings workflow (page 62)

• Verifying the configuration and user preferences (page 64)
• Running a performance check (page 67)
• Setting up an experiment (page 72)
• Creating application settings (page 77)
• Recording compensation controls (page 80)
• Calculating compensation (page 83)
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 Before you record data for a sample, optimize the cytometer
settings settings for the sample type and fluorochromes used.
Manual Compensation setup automatically calculates compensation

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 BD FACSCelesta flow cytometer and perform the setup
and QC procedures. See Cytometer setup (page 31).
Workflow for Cytometer optimization consists of the following steps.

optimizing settings
Step Description
1 Verifying the configuration and user preferences (page 64)
2 Running a performance check (page 67)
3 Setting up an experiment (page 72)

Step Description
4 Creating application settings (page 77)
5 Recording compensation controls (page 80)
6 Calculating compensation (page 83)
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 The examples in this chapter use a 4-color bead sample with the
examples 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.
Verifying the configuration and user preferences

Introduction This topic describes how to verify the cytometer configuration and
user preferences before you create an experiment.
Caution! To obtain accurate data results, the current

cytometer configuration must reflect your
BD FACSCelesta flow cytometer optics.
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.
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 FACSCelesta flow cytometer 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 67)
More information • Setting up an experiment (page 72)

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. If the baseline settings are not appropriate
for your applications, adjust the PMT voltage as necessary and use
the Application Settings to maintain Target Values.
Before you begin Define the performance baseline for any configuration before
running a performance check. See Custom configurations and
baselines (page 43).
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).
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 Fine Adjust is set to 250, 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.
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).
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 To create an experiment:

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.
5. Select MyExperiment in the Browser.
The Inspector displays details for the experiment.
Specifying To specify the parameters for the new experiment:

parameters 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

detector, you might have to select the one you need from a
menu. For example, you can set Detector D for the blue laser
as FITC or BB515.
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.
a. Click the selection button (to the left of the parameter

name) to select the parameter.
b. Click Delete.
The parameter is deleted.
Creating application settings

Introduction This topic describes how to create application settings.
About application Application settings are associated with a cytometer configuration

settings 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.
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.
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 appropriately for your sample
type.
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.
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 less than 1 mL 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 80)

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 To create compensation control tubes:

compensation 1. Select Experiment > Compensation Setup >
tubes 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.
Compensation control tubes are added to the experiment.

Worksheets containing appropriate plots and gates are added
for each compensation tube.
Recording To record compensation settings:

compensation 1. Press RUN and HIGH on the cytometer fluid control panel.
settings
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.
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.
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.
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.
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 83).
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.
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 medians of
the positive controls should be aligned with the medians of the
negative controls.
More information • Recording compensation controls (page 80)

6
Recording and analyzing data
• Data recording and analysis workflow (page 86)

• Preparing the workspace (page 87)
• Recording data (page 88)
• Analyzing data (page 91)
• Reusing an analysis (page 97)
Data recording and analysis workflow

Introduction This topic outlines the basic acquisition and analysis tasks using
BD FACSDiva software.
About the The examples in this chapter are from two 4-color bead samples
examples 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 61),
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 61).
Workflow for Recording and analyzing data consists of the following steps.
recording and
analyzing data
Step Description
1 Preparing the workspace (page 87)
2 Recording data (page 88)
3 Analyzing data (page 91)
4 Reusing an analysis (page 97)

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 When applications settings are applied to an experiment, the

application settings cytometer settings are updated with the parameters included in the
to a new application settings, optimized PMT voltages, threshold settings,
experiment 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.
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 77)

• 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.
Note: If you need to run samples at an event rate greater than

10,000 events/second, consider changing your Window extension.
See the BD FACSDiva Software Reference Manual for more
information.
Recording data To record data:

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.
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.
7. When event recording has completed, remove the first tube

from the cytometer.
The MyData worksheet plots should look like the following.
8. Install the second sample tube onto the SIP.
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.
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 with less than 1 mL of DI water onto the SIP.
17. Place the cytometer in standby mode.
More information • Analyzing data (page 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.
5. Draw a gate around the singlets on the FSC vs SSC plot.
6. Use the population hierarchy to rename the population

Singlets.
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.
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.
• 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.
e. Click OK.
12. Print the analysis.
Your global worksheet analysis objects should look like the

following.
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.
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 You can apply the global worksheet analysis to multiple tubes (on
analysis to normal a single normal worksheet) by selecting multiple tubes before
worksheets 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)

7
Technical overview
This chapter provides a technical overview of the following topics:
• About fluidics (page 100)

• About optics (page 101)
• About electronics (page 112)
About fluidics
Introduction This topic describes the BD FACSCelesta flow cytometer fluidics
system.
Pressure-driven The fluidics system in the BD FACSCelesta flow cytometer

fluidics system operates at a 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 The sample to be analyzed arrives in a separate pressurized stream.

focusing 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.
Low sample High sample

pressure pressure
Laser beam Laser beam
(12 µL/min) (60 µL/min)
Sheath Sheath Sheath Sheath

fluid Sample fluid fluid Sample fluid
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.
Changing the sample flow rate changes the sample pressure

thereby changing 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.
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).
Side scatter (SSC)
Light source Forward scatter (FSC)
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
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 128) for more information on
excitation and emission of fluorochromes. An interactive spectral
viewer is also available at bdbiosciences.com.
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.
Photons
absorbed
Photons Photons
transmitted 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 There are four types of filters.

filters
• Longpass (LP) filters. Transmit wavelengths at or longer than
the specified value.
• Shortpass (SP) filters. Transmit wavelengths at or shorter than

the specified value. This type of filter is not recommended.
• Bandpass (BP) filters. Pass a narrow spectral band of light by

combining the characteristics of shortpass filters, longpass
filters, and absorbing layers.
• 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 FACSCelesta uses LP filters and BP filters.
Longpass (LP) LP filters pass wavelengths longer than the filter rating. For
filters 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
% Transmission
Wavelength (nm)
Shortpass (SP) An SP filter has the opposite properties of an LP filter. An SP filter

filters passes light with a shorter wavelength than the filter rating. For
example, a 500-SP filter passes wavelengths of 500 nm or shorter,
and reflects or absorbs wavelengths longer than 500 nm.
Shortpass
% Transmission
Wavelength (nm)
Bandpass (BP) A BP filter transmits a relatively narrow range or band of light. BP

filters 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.
Bandpass
% Transmission
Wavelength (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.
% Transmission
Wavelength (nm)
Dichroic mirrors Dichroic filters that are used to direct different color light signals
to different detectors are called dichroic mirrors.
Although some of the properties of LP and SP filters are similar 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.
Compensation Fluorochromes emit light over a range of wavelengths. Optical

theory 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
yellow-green configuration will help minimize spillover.
Normalized intensity
Wavelength (nm)
This spillover can be seen in a dot plot of FITC vs PE. The FITC
spillover in the PE detector must be corrected as demonstrated in
the two figures that follow.
FITC-positive
PE
population
Unstained
particles
FITC
Using the Compensation tab of the Cytometer window in

BD FACSDiva software, you can adjust the PE-%FITC spectral
overlap value. Compensation is optimal when the positive and
negative FITC populations have the same medians in the PE
parameter statistics.
The following image shows the FITC spillover optimally

compensated out of the PE parameter.
FITC-positive
PE population
Unstained
particles
Matching medians
FITC
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.
FITC PE
Different intensity FITC signals

Normalized intensity
Same proportion or percentage of

spectral overlap in PE channel
Wavelength (nm)
About electronics
Introduction This topic describes the electronics in the BD FACSCelesta flow
cytometer
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.
Signal intensity
Time
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.
Signal intensity
Time
3. As the particle leaves the beam, the pulse trails off below the
threshold.
Signal intensity
Time
Pulse The pulse processors measure pulses by three characteristics:

measurements 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. The current width is measured by the electronics.

Digital electronics BD FACSCelesta 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 and area

• 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 and height 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)
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.
Note: 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 67)

8
Troubleshooting
• Cytometer troubleshooting (page 118)

• Electronics troubleshooting (page 126)
Cytometer troubleshooting
Introduction This topic describes possible problems and recommended solutions
for BD FACSCelesta flow cytometer 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 58).
Outer sleeve is not seated in 1. Loosen the retainer.

the retainer 2. Push the outer sleeve up into the
retainer until seated.
3. Tighten the retainer.
Outer sleeve is not on the Replace the outer sleeve.

sample injection tube
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, Check the waste line.

preventing proper aspiration
Waste tank is full Empty the waste tank.
Droplet containment vacuum Call your BD service representative.

is not functioning
The mode is not set to HTS. Change the mode to HTS by pressing
down the MODE button for more
than 3 seconds.
Sample tube not

fitting on SIP Possible causes Recommended solutions
Sample tube other than Use Falcon 12 x 75-mm polystyrene
Falcon® tubes used sample tubes. See Equipment
(page 146).
Worn Bal seal Replace the Bal seal. See Changing the
Bal seal (page 56).
Sample tube is cracked Transfer contents to a new tube.
Rapid sample
aspiration Possible causes Recommended solutions
Support arm is to the side Place the support arm under the
sample tube.
Droplet containment module Try the solutions in Droplets are

is failing visible on the SIP (page 118). If the
issue is not resolved, call your BD
service representative.
No events in
acquisition display Possible causes Recommended solutions
and RUN button is
Threshold is not set to the Set the threshold to the correct
green correct parameter (usually parameter for your application.
FSC)
Threshold level is too high Lower the threshold level.
PMT voltage for threshold Set the PMT voltage higher for the
parameter is set too low 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 35).
Possible causes Recommended solutions

Air bubble or debris in the Prime the fluidics system. See Priming
flow cell the fluidics (page 40).
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 Adjust the PMT voltages.

too high for display
parameter
Too few events are displayed Increase the number of events to

display.
Sample injection tube is Remove the sample tube to allow

clogged 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 56).
Instrument is not warmed up Wait 30 minutes for the instrument to

warm up.
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.

Waste tank is pressurized. Disconnect
the waste container for at least
30 seconds to allow the pressure to
dissipate.
No events in
acquisition display Possible causes Recommended solutions
and RUN button is
RUN is not activated Press the RUN button.
orange
Sample tube is not installed Install the sample tube correctly on the
or is not properly seated SIP.
Sample tube is cracked Replace the sample tube.
Waste tubing line is Connect the waste tubing line to the

disconnected from the waste waste container tank.
container tank
Sheath container is not  Ensure that the sheath container lid

pressurized and all connectors are securely
seated.
 Inspect the sheath container O-ring
inside the lid and replace it if
necessary. See Cleaning or
replacing the sheath gasket
(page 60)
Bal seal (page 56).
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 35).
No fluorescence
signal Possible causes Recommended solutions
Incorrect fluorochrome Make sure that the cytometer
assignment configuration in the software matches
the optical filters in the cytometer and
the configuration is as expected.
Laser is not functioning Call your BD service representative.

No signal in red
laser channels Possible causes Recommended solutions
(when red laser is
Incorrect laser delays due to  Check the fluid level in the sheath
installed) a change in the sheath tank tank and refill if necessary.
fluid level
High event rate

Air bubbles in the sheath Remove the air bubbles. See Removing
filter or flow cell air bubbles (page 35).
Threshold level is too low Increase the threshold level. See the
BD FACSDiva Software Reference
Manual for instructions.
PMT voltage for the Set the PMT voltage lower for the
threshold parameter is set threshold parameter. See the
too high BD FACSDiva Software Reference
Sample is too concentrated Dilute the sample.
Sample flow rate is set to Set the sample flow rate to MED or
HIGH LOW.
Low event rate

Threshold level is too high Lower the threshold level. See the
BD FACSDiva Software Reference
PMT voltage for the Set the PMT voltage higher for the
threshold parameter is set threshold parameter. See the
too low BD FACSDiva Software Reference

Sample is not adequately Mix the sample to suspend the cells.
mixed
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 HIGH.

Erratic event rate

Sample tube is cracked Replace the sample tube.
Bal seal (page 56).

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 54).
Distorted scatter
parameters Possible causes Recommended solutions
Cytometer settings are Optimize the scatter parameters. See
improperly adjusted the BD FACSDiva Software Reference
Air bubble in the sheath filter Purge the air from the filter. See
or flow cell Removing air bubbles (page 35).
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 Replace the buffers or fixative.

fixative
Excessive amount
of debris in display 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 54).

system (page 50).
Dead cells or debris in the Examine the sample under a

sample microscope.
Sample is contaminated Re-stain the sample. Ensure that the

tube is clean.
Stock sheath fluid is Rinse the sheath container with DI

contaminated water, then fill the container with
sheath fluid from another (or new lot)
bulk container.
High CV or poor QC
results Possible causes Recommended solutions
Air bubble in sheath filter or  Purge the filter. See Removing air
flow cell bubbles (page 35).
 Prime the fluidics system. See
Priming the fluidics (page 40).
Sample flow rate is set too Set the sample flow rate lower.
high
Air leak at sheath container Ensure that the sheath container lid is
tight and all connectors are secure.

system (page 50).
Waste tank is pressurized Replace the waste container cap. See

Replacing the waste container cap
(page 52).
Poor sample preparation Repeat sample preparation.
Sample was not diluted in the Dilute the sample in the same fluid as
same fluid as the sheath fluid you are using for sheath.
Old or contaminated QC Make new QC samples and perform

particles 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.

Electronics troubleshooting
Introduction This topic describes possible problems and recommended solutions
for BD FACSCelesta electronics issues.
“Cytometer
Disconnected” in Possible causes Recommended solutions
cytometer window
Cytometer power is off Turn on the cytometer main power.
Communication failure 1. In BD FACSDiva software, select

between workstation and Cytometer > Connect.
cytometer 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.
9
Detector array configurations
• Fluorescence spectra (page 128)

• About configuration maps (page 131)
• About the configuration (page 132)
• Base configuration polygon maps (page 134)
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 available at bdbiosciences.com.
You can also look for training by selecting Support > Training.
Designing The BD FACSCelesta flow cytometer is designed specifically for

multicolor panels multicolor research. 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 detectors. For more information about designing multicolor
panels, see Selecting Reagents for Multicolor Flow Cytometry
(Part No. 23-9538-02).
Example laser and The following figure shows the emission spectra of some common
dye interactions dyes, based on laser excitation. In many cases, a given dye can be
excited by multiple laser wavelengths, yielding different emission
intensities.
No Definition
1 UV (355 nm)
2 Violet (405 nm)
3 Blue (488 nm)
4 Yellow-Green (561 nm)
5 Red (640 nm)

About configuration maps

Introduction This topic describes the filter and mirror arrangements in the
detector arrays.
Filter and mirror The filters are arranged in the detector array to steer progressively
arrangement shorter wavelengths of light to the next detector 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.
About the configuration

Introduction This section describes the configuration options available with the
Four available The configuration for a BD FACSCelesta flow cytometer supports

configurations detectors, filters, and mirrors for up to three lasers to provide up to
12-color detection.
The following table explains the laser configuration set up in the

Optional Optional
Violet Violet Blue Blue third third
laser fluorescent laser fluorescent laser laser
Configuration emission detectors emission detectors emission detectors
2 laser BV 405 6 488 4 - -
3 laser BVUV 405 6 488 4 355 2
3 laser BVYG 405 6 488 2 571 4
3 laser BVR 405 5 488 4 640 3

Base configuration The BD FACSCelesta flow cytometer has one base configuration at
installation. The following image shows a default base cytometer
configuration.
More information • Verifying the configuration and user preferences (page 64)
• Base configuration polygon maps (page 134)
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 detector position.
Four-color blue The following map shows the four-color configuration for the
laser configuration 488-nm blue laser. This laser configuration is found in the BVR,
BVUV, and BV BD FACSCelesta flow cytometers.
Detector LP mirror BP filter Fluorochromes

A 670 695/40 PerCP-Cy5.5
B 600 610/20 PE-Texas Red®, PE-CF594
C 550 575/25 PE
D 505 530/30 FITC, BB515
E Blank 488/10 SSC
610/20
53
600LP 0/
30
50
5L
P
0
8/1
48
P
670LP 550 L
695 /25
/40 575
Six-color violet The following map shows the six-color configuration for the
laser configuration 405-nm violet laser. This laser configuration is found in the BVYG,
BVUV, and BV BD FACSCelesta flow cytometers.
PMT LP mirror BP filter Fluorochromes

A 750 780/60 BV786
B 690 710/50 BV711
C 655 670/30 BV650
D 595 610/20 BV605
E 505 525/50 BV510
F Blank 450/40 BV421
710/50
61
690LP 0/
20
59
5L
P
450/40
LP
0
505
5/5
52
P
750 LP 655L
780 0 /30
/60 67
Two-color ultra The following map shows the two-color configuration for the
violet laser 355-nm ultra violet laser. This laser configuration is found in the
configuration BVUV BD FACSCelesta flow cytometer.

A 690 740/35 BUV737
B Blank 379/28 BUV395
Five-color blue laser configuration
379/28
690LP
740
/35
Two-color blue The following map shows the two-color configuration for the
laser configuration 488-nm blue laser. This laser configuration is found in the BVYG

A 670 695/40 PerCP-Cy5.5
B 505 530/30 FITC, BB515
C Blank 488/10 SSC
530/30
505LP
670LP
695 /10
/40 488
.
Three-color red The following map shows the three-color configuration for the
laser configuration 640-nm red laser. This laser configuration is found in the BVR

A 750 780/60 APC-Cy7
B 690 730/45 APC-R700
C Blank 670/30 APC
730/45
690LP
750 LP
780 /30
/60 670
Four-color yellow- The following map shows the four-color configuration for the
green laser 571-nm yellow-green laser. This laser configuration is found in the
configuration BVYG BD FACSCelesta flow cytometer.

A 750 780/60 PE-Cy7
B 635 670/30 PE-Cy5
C 600 610/20 PE-Texas Red, PE-CF594
D Blank 586/15 PE
670/30
58
635LP 6/
15
P
750LP 60 0 L
780 /20
/60 610
Five-color violet The following map shows the five-color configuration for the
laser configuration 405-nm violet laser. This laser configuration is found in the BVR

A 750 780/60 BV786
B 655 670/30 BV650
C 595 610/20 BV605
D 505 525/50 BV510
E Blank 450/40 BV421
670/30
52
655 LP 5/
50
50
5L
P
0
0/4
45
P
750 LP 595L
780 /20
/60 610
More information • About the configuration (page 132)

10
Supplies and consumables
• Ordering information (page 144)

• Beads (page 144)
• Reagents (page 145)
• Equipment (page 146)
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 Blue, Violet, BD Biosciences 559123
Particles (8 peaks) Yellow-Green
and Red
SPHERO Ultra Rainbow Blue, Violet, Spherotech, Inc. URFP-30-2

Fluorescent Particles (single peak) Yellow-Green
and Red
BD DNA QC Particles Blue (488 nm) BD Biosciences 349523

Chapter 10: Supplies and consumables 145
CS&T beads
Bead Laser Supplier Catalog No.
BD FACSDiva 2.0  UV (355 nm) BD Biosciences  655050
CS&T research  Violet (405 nm) (50 tests)
beads  655051
 Blue (488 nm)
(150 tests)
 Red (640 nm)
 Yellow-green (561 nm)
Reagents
Reagent Supplier Catalog No.
BD FACSFlow sheath fluid BD Biosciences 342003
BD FACS™ sheath solution BD Biosciences 336524 (USA)

with surfactant (recommended
336911 (Europe)
for use with the HTS option)
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 BD Biosciences 660585

Concentrate
Reagent Supplier Catalog No.

BD FACSClean solution BD Biosciences 340345
Dyes and fluorochromes BD Biosciences, Life –

Technologies, or Sigma
Chlorine bleach Clorox® or other major –

(5% sodium hypochlorite) supplier (to ensure that the
bleach is at the correct
concentration and free of
particulate matter)
Equipment
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 bandpass (BP) filters
355-nm near-UV laser 137 detector array placement 29
405-nm violet laser 136, 141 theory 107
488-nm blue laser 135, 138 base configurations 134
571-nm yellow-green laser 140 BD Detergent Solution Concentrate 48
640-nm red laser 139 BD FACSClean solution 146
BD FACSDiva software See software
BD FACSFlow sheath fluid 145
A BD FACSFlow solution 35
acridine orange (AO) 47 BD FACSFlow supply system 32, 49
air bubbles, removing 35 BD High Throughput Sampler (HTS) 26
alarm blank optical holders 41
waste container 27 bleach 46, 146
analysis BP See bandpass filters
data 86 bubbles, removing air 36
immunophenotyping 91 buttons
reusing 97 fluid control 21
saving 97 sample flow rate control 22
antibodies 102
AO See acridine orange
C
application settings
applying 87 calculating compensation 83
creating 77 capacity, waste container 27
arm, tube support 25 cleaning solutions 46
aspiration (sample), rapid 119 compensation
assistance, technical 13 calculating 83
control tubes 80
recording settings 81
B theory 109
Bal seal components
ordering 146 sheath container shown 34
replacing 56 sheath filter shown 54
waste container shown 38 Diva software See software

computer system, about 30 DNA, flow rate for analysis 101
configuration droplet containment system 26
BD FACSDiva 64
containers E
sheath 27, 33
electronics
waste 27, 38, 40
digital 114
control panel, cytometer 19
laser controls 115
controls
pulse 112
fluidics 19
pulse measurements 113
single-stained 63, 86
threshold 114
conventions
troubleshooting 126
safety symbols 11
emission duration 112
creating
event rate
analysis objects 91
erratic 123
global worksheets 87
high 122
statistics view 91
low 122
CS&T particles 145
zero 119, 121
CV, troubleshooting 125
excessive debris 124
cytometer 17
excitation wavelength 102
configuration 42, 64
experiments
disconnect error 126
creating 72
setup 62
immunophenotyping 87
starting 32
sample optimization 72
troubleshooting 118
specifying parameters 73
D F
Daily cleaning 47
FACSFlow sheath fluid 145
DAPI, cleaning after using 47
FACSFlow solution 35
data
Falcon tubes 119
analyzing 86, 91
filters, optical
gating 91
bandpass (BP) 29, 107
recording 86, 88
dichroic See dichroic mirrors
debris, excessive 124
longpass (LP) 104, 105
detector arrays 28
notch 104
detectors 30
shortpass (SP) 104, 106
dichroic mirrors
theory 104
theory 108
filters, sheath
digital data 17
attaching new 55
digital electronics 114
Index 149
replacing 54 H
FITC and Stokes shift 103 hazard symbol definitions 11
fixed-alignment lasers 17 Help, accessing 11
flow cell High Throughput Sampler (HTS) 26
draining 40 hydrodynamic focusing 100
fluidics 100
flow rate control buttons 22
fluid control buttons I
PRIME 21 immunophenotyping
RUN 21 analysis 91
STANDBY 21 experiment 87
fluidics hydrodynamic focusing 101
description 20, 100
flow rate control 22 L
flushing system 50 lasers
priming 40 options 28
removing air bubbles 36 performance check 67
run mode 21 quality control (QC) particles 144
sample injection port (SIP) 25 longpass (LP) filters
fluidics controls, cytometer 19 defined 104
fluidics interconnect 37 theory 105
fluorescence LP See longpass filters
defined 102
emission 112
fluorescence signal, none 121 M
fluorochromes maintenance
compensation theory 109 Bal seal 56
emission spectra 103 cytometer shutdown 49
measured 42 sample tube O-ring 58
role in light emission 102 schedule 47
flushing the system 50 scheduled 50
forward scatter (FSC) 102 sheath filter 54
FSC See forward scatter system flush 50
mirrors, dichroic longpass (LP) filter 29
G
gating data 91 N
global worksheets notch filters 104
analyzing data 91
creating 87
previewing data 86, 97
O R
online Help 11 recording
optic holder 41 compensation settings 81
optics compensation tubes 80
components 28 data 86, 88
configuration 64, 66 removing air bubbles, filter 36
dichroic mirrors 108 replacing
filters 28, 104 Bal seal 56
optimization, sample 62 sample tube O-ring 58
ordering spare parts 144 sheath filter 54
O-ring waste air filter 52, 60
ordering 146 reusing analyses 97
replacing 58 RUN
worn 118 button orange 121
fluid control button 21
P
particles 144, 145 S
PerCP, Stokes shift 102 safety symbol definitions 11
performance check 67, 68 safety symbols 11
photodiode 30 sample
photomultiplier tubes (PMTs) 29 optimization 62
PMT See photomultiplier tubes optimization experiment 72
polygon optimization, single-stained
shown 29 controls 63, 86
population hierarchy 91 sample injection port (SIP)
PRIME, fluid control button 21 components 25
priming fluidics system 40 hydrodynamic focusing 100
propidium iodide (PI) 47 replacing Bal seal 56
pulse replacing sample tube O-ring 58
definition 112 sample tube
measurements 113 injection 25
processors 113 not fitting 119
requirements 119
Q samples, running 88
saving analyses 97
quality control (QC)
scatter
particles 144
light 102
troubleshooting 125
parameter distortion 124
setup, cytometer 62
Index 151
sheath requirements 119

flow 100
sheath container U
components shown 34
user preferences 66
defined 27
depressurize 34
preparing 34 W
sheath filter waste air filter
components shown 54 component shown 38
ordering 146 waste container 27
removing air bubbles 35 alarm 27
replacing 54 capacity 27
sheath fluid 100 components shown 38
BD FACSFlow sheath fluid 145 defined 27
shortpass (SP) filters 104, 106 emptying 38
side scatter (SSC) 102 workstation, about 30
signals, amplifying 29
SIP See sample injection port
software
cytometer control 10
version 30
spare parts, ordering 144
spillover 109
SSC See side scatter
STANDBY, fluid control button 21
starting cytometer 32
statistics views 91
Stokes shift 102
support, technical 13
T
technical assistance 13
thiazole orange (TO) 47
threshold, defined 114
TO See thiazole orange
troubleshooting
cytometer 118
electronics 126
tubes
Falcon 119

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For Research Use Only

Becton, Dickinson and Company BD Biosciences

SPHERO is a trademark of Spherotech, Inc.

Microsoft and Windows are registered trademarks of Microsoft Corporation.

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

Living Colors® (DsRed, mCherry) is a registered trademark of Clontech.

Falcon® is a registered trademark of Corning Incorporated.

CF™ is a trademark of Biotium, Inc.

Class 1 Laser Product.

Revision Date Change made

23-17147-00 11/2015 Initial release

23-17147-02 9/2020 Removed references to FACSRinse and added BD Detergent

Chapter 1: About this guide 9

Chapter 3: Cytometer setup 31

Chapter 5: Optimizing cytometer settings 61

Chapter 6: Recording and analyzing data 85

Chapter 7: Technical overview 99

Chapter 8: Troubleshooting 117

Chapter 9: Detector array configurations 127

Chapter 10: Supplies and consumables 143

• What this guide covers (page 10)

What this guide covers

This guide assumes you have a working knowledge of basic

About the BD FACSCelesta documentation

Publication formats This guide is provided in PDF format to provide an eco-friendly

BD FACSDiva software. Internet access is not required to use the

The help system is compiled from the following documents:

• BD FACSDiva Software Reference Manual: Includes

Additional BD Cytometer Setup and Tracking Application Guide: Describes

Instrument technical support

Contacting If technical assistance is required, contact your local

When contacting BD Biosciences, have the following information

• Product name, part number, and serial number

2. Select your region. You will see information in your local

• System overview (page 16)

2 BD FACSCelesta flow cytometer

Components The following figure shows the main components of the

5 Fluidic sensor ports

6 Sample injection port (SIP)

7 Heat ventilation slots (left side)

8 Air and fluidic ports

9 Optics access door (polygon

Caution! Do not place any objects on top of the

Caution: Electrical Hazard! Do not place liquids on top of

2 Fluid control buttons

3 Sample flow rate buttons

More information • Fluidics system (page 20)

LED color Status Action Condition

Yellow Caution! Sheath Check tank levels Sheath low and/or

Red Caution! Take  Empty waste tank Sheath empty and/or

System status is also displayed on the Status screen. See Status

• Activity. Shows whether the cytometer power is on and the

Indicator LED color Status

Fluctuates blue Cells are passing through the

• RUN. Pressurizes the sample tube to transport the sample

• STANDBY. Stops fluid flow to conserve sheath fluid.

• PRIME. Prepares the fluidics system by draining and filling the

Note: Remove sample tubes during the prime cycle.