BD FACSCelesta Flow Cytometer User Manual
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BD FACSCelesta Flow Cytometer User Manual

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For Research Use Only
23-17147-02
9/2020
Becton, Dickinson and Company
BD Biosciences
2350 Qume Drive
San Jose, CA 95131 USA
.
bdbiosciences
com
ResearchApplications@bd.com
BD FACSCelesta™ Flow
Cytometer
User's Guide
BD Biosciences
European Customer Support
Tel +32.53.720.600
help.biosciences@bd.com

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  • Page 1 BD FACSCelesta™ Flow Cytometer User’s Guide For Research Use Only 23-17147-02 9/2020 Becton, Dickinson and Company BD Biosciences European Customer Support BD Biosciences Tel +32.53.720.600 2350 Qume Drive help.biosciences@bd.com San Jose, CA 95131 USA bdbiosciences ResearchApplications@bd.com...
  • Page 2 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.
  • Page 3 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.

  • Page 5: Table Of Contents

    Conventions ........... . 11 About the BD FACSCelesta documentation ......11 Instrument technical support .
  • Page 6 BD FACSCelesta Flow Cytometer User’s Guide Chapter 4: Maintenance Maintenance overview ..........46 Cleaning the fluidics .
  • Page 7 Contents Chapter 8: Troubleshooting Cytometer troubleshooting ........118 Electronics troubleshooting .

  • Page 9: Chapter 1: About This Guide

    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)

  • Page 10: What This Guide Covers

    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.

  • Page 11: Conventions

    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.
  • Page 12 BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 13: Instrument Technical Support

    Introduction 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 •...
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  • Page 15: Chapter 2: Introduction

    Introduction This chapter covers the following topics: • System overview (page 16) • Control panel (page 19) • Fluidics system (page 20) • Sheath and waste containers (page 27) • Optics (page 28) • Workstation (page 30)

  • Page 16: System Overview

    BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 17: Cytometer Overview

    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.
  • Page 18 BD FACSCelesta Flow Cytometer User’s Guide Number Component Heat ventilation slots (top) Control panel Power button Electrical plug Fluidic sensor ports Sample injection port (SIP) Heat ventilation slots (left side) Air and fluidic ports Optics access door (polygon detector arrays)

  • Page 19: Control Panel

    Chapter 2: Introduction 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.

  • Page 20: Fluidics System

    BD FACSCelesta Flow Cytometer User’s Guide Number Component Sample fine adjust buttons Status screen 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.
  • Page 21 Chapter 2: Introduction 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.
  • Page 22 BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 23 Chapter 2: Introduction The following table shows the approximate sample flow rate range for low, medium, and high. Settings Sample flow rate (µL/min) 6–24 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.
  • Page 24 BD FACSCelesta Flow Cytometer User’s Guide Line Definition 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.
  • Page 25 Chapter 2: Introduction Sample injection The SIP is where the sample tube is installed. The SIP includes the sample injection tube and the tube support arm. Samples are port (SIP) 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.
  • Page 26 BD FACSCelesta Flow Cytometer User’s Guide Cautions when Caution: Biohazard! When using the BD FACSCelesta using the HTS flow cytometer with the HTS, ensure that the HTS is option completely pushed into the operating position before removing the droplet containment module (DCM) sleeve or disconnecting the sample coupler from the SIP.

  • Page 27: Sheath And Waste Containers

    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. The sheath container has a capacity of 10 L. Sheath fluid is filtered...

  • Page 28: Optics

    Laser options The BD FACSCelesta flow cytometer can be configured with up to three lasers as listed in the following table. Laser...
  • Page 29 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.

  • Page 30: Workstation

    This topic describes the components of the BD FACSCelesta workstation. Acquisition, analysis, and most instrument functions are controlled Workstation 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 •...

  • Page 31: Chapter 3: Cytometer Setup

    Cytometer setup This chapter covers the following topics: • 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) •...

  • Page 32: 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. To start the cytometer: Procedure 1.

  • Page 33: Preparing The Sheath Container

    This topic describes how to prepare the sheath container. Introduction Note: If your system is using the BD FACSFlow supply system, see the documentation provided with your system. Check the fluid levels in the sheath container every time you use When to check the the cytometer.
  • Page 34 BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 35: Removing Air Bubbles

    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.
  • Page 36 BD FACSCelesta Flow Cytometer User’s Guide To remove air bubbles: Procedure 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.
  • Page 37 Chapter 3: Cytometer setup 3. Direct the vent line into a beaker and press the small button at the end of the vent fitting against the side of the beaker until a steady stream of fluid empties from the filter. Button Vent fitting 4.

  • Page 38: Preparing The Waste Container

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 39 Chapter 3: Cytometer setup 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. •...

  • Page 40: Priming The Fluidics

    BD FACSCelesta Flow Cytometer User’s Guide 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...

  • Page 41: About The Optical Filters And Mirrors

    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.
  • Page 42 BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 43: Custom Configurations And Baselines

    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.
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  • Page 45: Chapter 4: Maintenance

    Maintenance This chapter covers the following topics: • Maintenance overview (page 46) • Cleaning the fluidics (page 47) • Shutting down the cytometer (page 49) • Flushing the system (page 50) • Replacing the waste container cap (page 52) • Changing the sheath filter (page 54) •...

  • Page 46: Maintenance Overview

    BD FACSCelesta Flow Cytometer User’s Guide 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 and materials can transmit potentially fatal disease.

  • Page 47: Cleaning The Fluidics

    Chapter 4: Maintenance 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)
  • Page 48 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.

  • Page 49: Shutting Down The Cytometer

    Chapter 4: Maintenance Shutting down the cytometer This topic describes how to shut down the cytometer. Introduction 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.

  • Page 50: Flushing The System

    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.
  • Page 51 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. Maintenance overview (page 46) for other recommended cleaning solutions.

  • Page 52: Replacing The Waste Container Cap

    BD FACSCelesta Flow Cytometer User’s Guide 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 and materials can transmit potentially fatal disease.
  • Page 53 Chapter 4: Maintenance 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.

  • Page 54: Changing The Sheath Filter

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 55 Chapter 4: Maintenance 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: •...

  • Page 56: Changing The Bal Seal

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 57 Chapter 4: Maintenance 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.

  • Page 58: Changing The Sample Tube O-Ring

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 59 Chapter 4: Maintenance 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.

  • Page 60: Cleaning Or Replacing The Sheath Gasket

    BD FACSCelesta Flow Cytometer User’s Guide Cleaning or replacing the sheath gasket Introduction This topic describes how to clean or replace the gasket of the sheath tank lid. We recommend cleaning the sheath gasket when needed. When to change the sheath gasket...

  • Page 61: Chapter 5: Optimizing Cytometer Settings

    Optimizing cytometer settings This chapter covers the following topics: • 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) •...

  • Page 62: Cytometer Settings Workflow

    For additional details, see the BD FACSDiva Software Reference Manual. Start the BD FACSCelesta flow cytometer and perform the setup Before you begin and QC procedures. See Cytometer setup (page 31).
  • Page 63 Chapter 5: Optimizing cytometer settings Step Description Creating application settings (page 77) Recording compensation controls (page 80) 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.

  • Page 64: Verifying The Configuration And User Preferences

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 65 Chapter 5: Optimizing cytometer settings Your cytometer might include only the base configuration when your cytometer is installed. You can create additional configurations later as needed.
  • Page 66 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 •...

  • Page 67: Running A Performance Check

    Chapter 5: Optimizing cytometer settings Running a performance check This topic describes how to run a performance check as part of Introduction 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...
  • Page 68 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).
  • Page 69 Chapter 5: Optimizing cytometer settings 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.
  • Page 70 BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 71 Chapter 5: Optimizing cytometer 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. Continue the optimization of your cytometer for an experiment or Next step sample type as described in Setting up an experiment (page...

  • Page 72: Setting Up An Experiment

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 73 Chapter 5: Optimizing cytometer settings 5. Select MyExperiment in the Browser. The Inspector displays details for the experiment. To specify the parameters for the new experiment: Specifying parameters 1. Select Cytometer Settings for the experiment in the Browser. Cytometer settings appear in the Inspector.
  • Page 74 BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 75 Chapter 5: Optimizing cytometer settings 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.
  • Page 76 BD FACSCelesta Flow Cytometer User’s Guide a. Click the selection button (to the left of the parameter name) to select the parameter. b. Click Delete. The parameter is deleted.

  • Page 77: Creating Application Settings

    Chapter 5: Optimizing cytometer settings Creating application settings Introduction This topic describes how to create application settings. About application Application settings are associated with a cytometer configuration and include the parameters for the application, area scaling values, settings 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.
  • Page 78 BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 79 Chapter 5: Optimizing cytometer settings 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.

  • Page 80: Recording Compensation Controls

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 81 Chapter 5: Optimizing cytometer settings 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.
  • Page 82 BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 83: Calculating Compensation

    Chapter 5: Optimizing cytometer settings 13. Verify that the snap-to interval gate encompasses the positive population. 14. Repeat steps for the remaining compensation tubes. After you have recorded data for each single-stained control, Next step calculate compensation as described in Calculating compensation (page 83).
  • Page 84 BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 85: Chapter 6: Recording And Analyzing Data

    Recording and analyzing data This chapter covers the following topics: • Data recording and analysis workflow (page 86) • Preparing the workspace (page 87) • Recording data (page 88) • Analyzing data (page 91) • Reusing an analysis (page 97)

  • Page 86: Data Recording And Analysis Workflow

    BD FACSCelesta Flow Cytometer User’s Guide Data recording and analysis workflow Introduction This topic outlines the basic acquisition and analysis tasks using BD FACSDiva software. The examples in this chapter are from two 4-color bead samples About the examples with the following fluorochromes: •...

  • Page 87: Preparing The Workspace

    Chapter 6: Recording and analyzing data Preparing the workspace This topic describes how to prepare the workspace and apply Introduction 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.

  • Page 88: Recording Data

    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: 1.
  • Page 89 Chapter 6: Recording and analyzing data 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.
  • Page 90 BD FACSCelesta Flow Cytometer User’s Guide The MyData worksheet plots should look like the following. 8. Install the second sample tube onto the SIP. 9. Set the current tube pointer to Beads_002. 10. Click Acquire Data to begin acquisition. 11. Before recording, preview the data on the MyData worksheet to verify that all expected populations are visible and the data is similar to the previous sample.

  • Page 91: Analyzing Data

    Chapter 6: Recording and analyzing data 16. Install a tube with less than 1 mL of DI water onto the SIP. 17. Place the cytometer in standby mode. • Analyzing data (page 91) More information 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.
  • Page 92 BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 93 Chapter 6: Recording and analyzing data 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.
  • Page 94 BD FACSCelesta Flow Cytometer User’s Guide • In the FITC vs PE plot, draw a gate around the FITC-positive population. Name the population FITC positive in the population hierarchy. • In the FITC vs PE plot, draw a gate around the PE-positive population.
  • Page 95 Chapter 6: Recording and analyzing data e. Click OK. 12. Print the analysis.
  • Page 96 BD FACSCelesta Flow Cytometer User’s Guide Your global worksheet analysis objects should look like the following.

  • Page 97: Reusing An Analysis

    Chapter 6: Recording and analyzing data More information • Reusing an analysis (page 97) Reusing an analysis Introduction This topic describes how to use a global worksheets to apply 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.
  • Page 98 BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 99: Chapter 7: Technical Overview

    Technical overview This chapter provides a technical overview of the following topics: • About fluidics (page 100) • About optics (page 101) • About electronics (page 112)

  • Page 100: About Fluidics

    BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 101: About Optics

    Chapter 7: Technical overview 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.
  • Page 102 BD FACSCelesta Flow Cytometer User’s Guide When a cell or particle passes through a focused laser beam, laser Light scatter 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).
  • Page 103 Chapter 7: Technical overview FITC have a smaller Stokes shift, absorbing blue light (488 nm) and emitting green light (530 nm). The following figure shows the emission spectra of some commonly used fluorochromes. Actual emission intensity will depend on excitation wavelength. Fluorescence spectra (page 128) for more information on excitation and emission of fluorochromes.
  • Page 104 BD FACSCelesta Flow Cytometer User’s Guide 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...
  • Page 105 Chapter 7: Technical overview 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.
  • Page 106 BD FACSCelesta Flow Cytometer User’s Guide An SP filter has the opposite properties of an LP filter. An SP filter Shortpass (SP) 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.
  • Page 107 Chapter 7: Technical overview A BP filter transmits a relatively narrow range or band of light. BP Bandpass (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.
  • Page 108 BD FACSCelesta Flow Cytometer User’s Guide Wavelength (nm) Dichroic filters that are used to direct different color light signals Dichroic mirrors to different detectors are called dichroic mirrors. Although some of the properties of LP and SP filters are similar to...
  • Page 109 Chapter 7: Technical overview Fluorochromes emit light over a range of wavelengths. Optical Compensation 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.
  • Page 110 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.
  • Page 111 Chapter 7: Technical overview FITC Different intensity FITC signals Same proportion or percentage of spectral overlap in PE channel Wavelength (nm)

  • Page 112: About Electronics

    BD FACSCelesta Flow Cytometer User’s Guide 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...
  • Page 113 Chapter 7: Technical overview 3. As the particle leaves the beam, the pulse trails off below the threshold. Time Pulse The pulse processors measure pulses by three characteristics: height, area, and width. measurements • Height. The maximum digitized intensity measured for the pulse.
  • Page 114 BD FACSCelesta Flow Cytometer User’s Guide BD FACSCelesta electronics digitize the signal intensity produced Digital electronics by a detector. The digitized data is stored in memory and further processed by the electronics to calculate: • Pulse height and area •...
  • Page 115 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 •...
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  • Page 117: Chapter 8: Troubleshooting

    Troubleshooting This chapter covers the following topics: • Cytometer troubleshooting (page 118) • Electronics troubleshooting (page 126)

  • Page 118: Cytometer Troubleshooting

    BD FACSCelesta Flow Cytometer User’s Guide 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...
  • Page 119 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...
  • Page 120 BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 121 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.
  • Page 122 BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 123 Chapter 8: Troubleshooting Possible causes Recommended solutions 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.
  • Page 124 BD FACSCelesta Flow Cytometer User’s Guide Distorted scatter parameters Possible causes Recommended solutions Cytometer settings are Optimize the scatter parameters. See improperly adjusted the BD FACSDiva Software Reference Manual for instructions. Air bubble in the sheath filter Purge the air from the filter. See...
  • Page 125 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.

  • Page 126: Electronics Troubleshooting

    BD FACSCelesta Flow Cytometer User’s Guide 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.

  • Page 127: Chapter 9: Detector Array Configurations

    Detector array configurations This chapter covers the following topics: • Fluorescence spectra (page 128) • About configuration maps (page 131) • About the configuration (page 132) • Base configuration polygon maps (page 134)

  • Page 128: Fluorescence Spectra

    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. The BD FACSCelesta flow cytometer is designed specifically for Designing multicolor panels multicolor research. Results depend on the excitation and emission...
  • Page 129 Chapter 9: Detector array configurations The following figure shows the emission spectra of some common Example laser and dye interactions dyes, based on laser excitation. In many cases, a given dye can be excited by multiple laser wavelengths, yielding different emission intensities.
  • Page 130 BD FACSCelesta Flow Cytometer User’s Guide Definition UV (355 nm) Violet (405 nm) Blue (488 nm) Yellow-Green (561 nm) Red (640 nm)

  • Page 131: About Configuration Maps

    Chapter 9: Detector array configurations About configuration maps Introduction This topic describes the filter and mirror arrangements in the detector arrays. The filters are arranged in the detector array to steer progressively Filter and mirror 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.

  • Page 132: About The Configuration

    This section describes the configuration options available with the BD FACSCelesta flow cytometer. 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.
  • Page 133 Chapter 9: Detector array configurations The BD FACSCelesta flow cytometer has one base configuration at Base configuration 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)

  • Page 134: Base Configuration Polygon Maps

    BD FACSCelesta Flow Cytometer User’s Guide 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.
  • Page 135 Chapter 9: Detector array configurations The following map shows the four-color configuration for the Four-color blue 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 695/40 PerCP-Cy5.5...
  • Page 136 BD FACSCelesta Flow Cytometer User’s Guide The following map shows the six-color configuration for the Six-color violet laser configuration 405-nm violet laser. This laser configuration is found in the BVYG, BVUV, and BV BD FACSCelesta flow cytometers. LP mirror BP filter...
  • Page 137 Chapter 9: Detector array configurations The following map shows the two-color configuration for the Two-color ultra violet laser 355-nm ultra violet laser. This laser configuration is found in the configuration BVUV BD FACSCelesta flow cytometer. LP mirror BP filter Fluorochromes 740/35 BUV737...
  • Page 138 BD FACSCelesta Flow Cytometer User’s Guide The following map shows the two-color configuration for the Two-color blue laser configuration 488-nm blue laser. This laser configuration is found in the BVYG BD FACSCelesta flow cytometer. LP mirror BP filter Fluorochromes 695/40 PerCP-Cy5.5...
  • Page 139 Chapter 9: Detector array configurations The following map shows the three-color configuration for the Three-color red laser configuration 640-nm red laser. This laser configuration is found in the BVR BD FACSCelesta flow cytometer. LP mirror BP filter Fluorochromes 780/60 APC-Cy7...
  • Page 140 BD FACSCelesta Flow Cytometer User’s Guide The following map shows the four-color configuration for the Four-color yellow- green laser 571-nm yellow-green laser. This laser configuration is found in the configuration BVYG BD FACSCelesta flow cytometer. LP mirror BP filter Fluorochromes...
  • Page 141 Chapter 9: Detector array configurations The following map shows the five-color configuration for the Five-color violet laser configuration 405-nm violet laser. This laser configuration is found in the BVR BD FACSCelesta flow cytometer. LP mirror BP filter Fluorochromes 780/60 BV786...
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  • Page 143: Chapter 10: Supplies And Consumables

    Supplies and consumables This chapter covers the following topics: • Ordering information (page 144) • Beads (page 144) • Reagents (page 145) • Equipment (page 146)

  • Page 144: Ordering Information

    BD FACSCelesta Flow Cytometer User’s Guide Ordering information To order spare parts and consumables from BD Biosciences: • Within the US, call (877) 232-8995. • Outside the US, contact your local BD Biosciences customer support representative. Worldwide contact information can be found at bdbiosciences.com.

  • Page 145: Reagents

    (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)

  • Page 146: Equipment

    BD FACSCelesta Flow Cytometer User’s Guide 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...
  • Page 147 640-nm red laser 139 BD FACSDiva software See software BD FACSFlow sheath fluid 145 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...
  • Page 148 BD FACSCelesta Flow Cytometer User’s Guide 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 sheath 27, 33 electronics waste 27, 38, 40...
  • Page 149 Index replacing 54 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 immunophenotyping PRIME 21 analysis 91...
  • Page 150 BD FACSCelesta Flow Cytometer User’s Guide 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...
  • Page 151 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...
  • Page 152 BD FACSCelesta Flow Cytometer User’s Guide...

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