BD FACSymphony A5 SE User Manual
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BD FACSymphony A5 SE User Manual

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BD FACSymphony™ A5 SE
Flow Cytometer
User's Guide
23-23473(02)
2022-08
English
 For Research Use Only

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Summary of Contents for BD FACSymphony A5 SE

  • Page 1: Bd Facsymphony™ A5 Se

    BD FACSymphony™ A5 SE Flow Cytometer User's Guide 23-23473(02) 2022-08  For Research Use Only English...
  • Page 2 BD. The information in this guide is subject to change without notice. BD reserves the right to change its products and services at any time. Although this guide has been prepared with every precaution to ensure accuracy, BD assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information.

  • Page 3: Table Of Contents

    Contents BD FACSYMPHONY™ A5 SE 1 ABOUT THIS GUIDE What this guide covers Conventions About the documentation Instrument technical support 2 INTRODUCTION System overview Cytometer overview Control panel Fluidics system Sheath and waste containers Optics Workstation 3 CYTOMETER SETUP Starting the cytometer and computer...
  • Page 4 BD FACSymphony™ A5 SE User's Guide Replacing the waste container cap Changing the sheath filter Changing the Bal seal Changing the sample tube O-ring Cleaning or replacing the sheath gasket 5 SPECTRAL FUNCTIONS Spectral function overview Autofluorescence Spectral unmixing software considerations...
  • Page 5 Contents Equipment INDEX...
  • Page 6 BD FACSymphony™ A5 SE User's Guide...

  • Page 7: About This Guide

    About this guide This chapter covers the following topics: What this guide covers (page 8) Conventions (page 8) About the documentation (page 9) Instrument technical support (page 10)

  • Page 8: What This Guide Covers

    BD FACSymphony™ A5 SE User's Guide What this guide covers This guide describes the procedures necessary to operate and maintain the BD FACSymphony™ A5 SE Special Order Research Product (SORP) 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 9: About The Documentation

     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.

  • Page 10: Instrument Technical Support

    Instrument technical support Introduction This topic describes how to get technical assistance. Contacting technical support If technical assistance is required, contact your local BD Biosciences customer support representative or supplier. Go to our website bdbiosciences.com for up-to-date contact information. When contacting BD Biosciences, have the following information available: Product name, part number, and serial number Version of BD FACSDiva™...

  • Page 11: Introduction

    Introduction This chapter covers the following topics: System overview (page 12) Cytometer overview (page 12) Control panel (page 14) Fluidics system (page 15) Sheath and waste containers (page 19) Optics (page 20) Workstation (page 22)

  • Page 12: System Overview

    System overview About the system The BD FACSymphony™ A5 SE system includes the flow cytometer, BD FACSDiva™ software v9.3 or later (for Windows 10) 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 13 Chapter 2  Introduction Components The following figure shows the main components of the instrument. Each component is described in detail in the following sections. Number Component Number Component Heat ventilation slots (top) Sample injection port (SIP) Control panel Heat ventilation slots (side) Power button Air and fluidic ports Electrical plug...

  • Page 14: Control Panel

    BD FACSymphony™ A5 SE User's Guide Control panel Overview The following figure shows the components in the control panel, which are listed in the table. Number Component System indicators Fluid control buttons Sample flow rate buttons Sample fine adjust buttons...

  • Page 15: Fluidics System

    Chapter 2  Introduction Fluidics system Introduction The fluidics system carries the sample out of the sample tube and into the sensing region of the flow cell. Cells are carried in the sample core stream in single file and measured individually. System indicators There are two system indicators (System status and Activity) on the control panel.
  • Page 16 BD FACSymphony™ A5 SE User's Guide Sample flow rate control The three flow rate control buttons (LOW, MED, HIGH) set the sample flow rate through the flow cell. The SAMPLE ADJ buttons allow you to adjust the rate to intermediate levels.
  • Page 17 Chapter 2  Introduction Item Definition Waste level. Shows range (1) 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. Sheath level. Shows range from E to F. The display line decreases from right to left in sequences of 20% from full level.
  • Page 18 BD FACSymphony™ A5 SE User's Guide Description Tube support arm Outer sleeve Sample injection tube Sample injection tube Stainless steel tube that carries sample from the sample tube to the flow cell. This tube is covered with an outer sleeve that serves as part of the droplet containment system.

  • Page 19: Sheath And Waste Containers

    The sheath and waste containers should remain in the installed position. Moving them to a different height (for example, from the floor to a bench) changes the flow rate. Note: If you are using the FFSS, see the BD FACSFlow™ Supply System User’s Guide. Sheath container The sheath container has a capacity of 10 L.

  • Page 20: Optics

    BD FACSymphony™ A5 SE User's Guide Optics Introduction This topic describes the optical components for the BD FACSymphony™ A5 SE flow cytometer including: Detector arrays Laser options Optical filters Signal detectors Detector arrays The detector arrays consist of cascadagon arrays. Each cascadagon is outfitted with 2 to 20 PMTs and can detect the corresponding number of signals.
  • Page 21 This base configuration is factory set. Cytometer configuration in BD FACSDiva™ BD FACSDiva™ software provides a base configuration for your flow cytometer. In the case of the BD FACSymphony™ A5 SE flow cytometer, the base configuration corresponds to the fixed configuration for the spectral function.

  • Page 22: Workstation

    Your workstation is equipped with the following: Microsoft Windows operating system BD FACSDiva™ software version 9.3 or later for Windows 10 for data acquisition and analysis Software documentation including the help system accessible from a menu in the BD FACSDiva™ software.

  • Page 23: Cytometer Setup

    Cytometer setup This chapter covers the following topics: Starting the cytometer and computer (page 24) Preparing the sheath container (page 25) Removing air bubbles (page 27) Preparing the waste container (page 29) Preparing the fluidics (page 31) Adding a bead lot number (page 32) Custom configurations and baselines (page 34)

  • Page 24: Starting The Cytometer And Computer

    Failure to warm up and stabilize the instrument could affect sample data. 5. Start BD FACSDiva™ software by double-clicking the shortcut on the desktop, and log in to the software. 6. Check the Cytometer window in BD FACSDiva™ software to ensure that the cytometer is connected to the workstation.

  • Page 25: Preparing The Sheath Container

    Introduction This topic describes how to prepare the sheath container. Note: If your system is using the FFSS, see the BD FACSFlow™ Supply System User’s Guide. When to check the sheath and waste containers Check the fluid levels in the sheath container and waste container every time you use the cytometer. This ensures that you do not run out of sheath fluid during an experiment and that the waste container does not overflow.
  • Page 26 BD FACSymphony™ A5 SE User's Guide Sheath container components The sheath container components are shown in the following image: Item Number Component Cap handle Filter assembly Sheath fluid line (blue) to flow cytometer Air line (green) Alarm sensor Clamp knob...

  • Page 27: 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. 5. Add 10 L of sheath fluid, such as BD FACSFlow™ solution, to the sheath container. Note: The 10 L will reach the interior line on the sheath tank. Do not fill the sheath tank further.
  • Page 28 BD FACSymphony™ A5 SE User's Guide Procedure To remove air bubbles: 1. Check the sheath filter for trapped air bubbles. Item Number Component Flow 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 29: Preparing The Waste Container

    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. Item Number Component Button Vent fitting...
  • Page 30 BD FACSymphony™ A5 SE User's Guide Waste container components The following figure shows the main components of the waste container. Item Number Component Baffle User-replacable cap Air line Alarm sensor Waste fluid line with clip Contact with biological specimens and materials can transmit potentially fatal disease.

  • Page 31: Preparing The Fluidics

    1. Turn on the computer and instrument as described in Starting the cytometer and computer (page 24). ® 2. Install a tube with 3 mL of 1.5% dilution of BD Detergent Solution Concentrate on the SIP and put the tube support arm underneath the tube. ®...

  • Page 32: Adding A Bead Lot Number

    Do not import a bead lot file as described in the BD  Cytometer Setup and Tracking Application Guide as the standard bead lot files are not applicable to the BD FACSymphony™ A5 SE flow cytometer. Use the following procedure to add a bead lot number instead.
  • Page 33 Chapter 3  Cytometer setup 6. Enter the expiration date. 7. Click OK.

  • Page 34: Custom Configurations And Baselines

    Optimizing cytometer settings (page 67). 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 about creating custom configurations and defining a baseline. More information...

  • Page 35: Maintenance

    Maintenance This chapter covers the following topics: Maintenance overview (page 36) Cleaning the fluidics (page 37) Shutting down the cytometer (page 38) Flushing the system (page 38) Replacing the waste container cap (page 40) Changing the sheath filter (page 41) Changing the Bal seal (page 42) Changing the sample tube O-ring (page 44) Cleaning or replacing the sheath gasket (page 45)

  • Page 36: Maintenance Overview

    BD FACSymphony™ A5 SE User's Guide Maintenance overview Introduction This topic provides an overview of the flow cytometer routine maintenance and cleaning procedures. General use guidelines Contact with biological specimens and materials can transmit potentially fatal disease. Follow these guidelines whenever operating or maintaining the cytometer: Wear suitable protective clothing, eyewear, and gloves.

  • Page 37: Cleaning The Fluidics

    3. Move the tube support arm under the tube (vacuum off) and allow the cleaning solution to run for 5 minutes with the sample flow rate set to HIGH. 4. Repeat steps 2 and 3 with DI water. ® 5. Repeat steps 2 and 3 with 1.5% dilution of BD Detergent Solution Concentrate. ® Note: The BD Detergent Solution Concentrate must be diluted before use.

  • Page 38: Shutting Down The Cytometer

    Note: After installation of a new system or major repair of the fluidics system, perform a system flush with overnight soak twice a week for one month. Note: If you are using the FFSS, see the BD FACSFlow™ Supply System User’s Guide for instructions on flushing the system.
  • Page 39 Press the PRIME button on the fluidics control panel. b. When the STANDBY button lights (amber), press the PRIME button again. 8. Install a tube with 3 mL of undiluted BD FACSClean™ solution on the SIP and put the tube support arm underneath the tube.

  • Page 40: Replacing The Waste Container Cap

    BD FACSymphony™ A5 SE 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. 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 41: Changing The Sheath Filter

    Chapter 4  Maintenance Changing the sheath filter Introduction This topic describes how to change the sheath filter. The sheath filter is connected in-line with the sheath line. It filters the sheath fluid as it comes from the sheath container. When to change the sheath filter We recommend changing the sheath filter assembly every six months.

  • Page 42: Changing The Bal Seal

    BD FACSymphony™ A5 SE User's Guide Removing the old filter To remove the old filter: 1. Place the cytometer in standby mode. 2. Remove the sheath filter assembly by pressing the quick-disconnect on both sides of the filter assembly. 3. Over a sink or beaker: Remove the vent line from the filter and set it aside.
  • Page 43 Chapter 4  Maintenance Procedure To replace the Bal seal: 1. Remove the outer sleeve from the sample injection tube by turning the retainer counterclockwise. Slide the outer sleeve down and off of the sample injection tube. Item Number Component Bal seal Retainer Outer sleeve Sample injection tube...

  • Page 44: Changing The Sample Tube O-Ring

    BD FACSymphony™ A5 SE User's Guide 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.

  • Page 45: Cleaning Or Replacing The Sheath Gasket

    Chapter 4  Maintenance 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.
  • Page 46 BD FACSymphony™ A5 SE User's Guide...

  • Page 47: Spectral Functions

    Spectral functions This chapter covers the following topics: • Spectral function overview (page 48) • Autofluorescence (page 54) • Spectral unmixing software considerations (page 55) • Setting up a new spectral experiment (page 57) • Creating a Spectral Unmixing matrix (page 59) •...

  • Page 48: Spectral Function Overview

    The software for the instrument, BD FACSDiva™ v9.3 or later, supports customer workflows that can be either spectral or traditional compensation experiments. This section describes the spectral functionality and workflows.
  • Page 49 Note: The preceding diagram shows the UV laser emission frequency at 355 nm. An alternative laser is available with an emission frequency of 349 nm. The detectors and filter set used with the laser are the same in each case. The BD FACSymphony™ A5 SE achieves the illustrated spectral responses using the following laser /detector/filter combinations:...
  • Page 50 BD FACSymphony™ A5 SE User's Guide Laser Detector Channel Mirror Filter Parameter Blue (488 nm) 770 LP 810/79 BP B810 724 LP 750/60 BP B750 685 LP 710/50 BP B710 675/20 BP B675 665 LP 645 LP 660/30 BP B660 585 LP...
  • Page 51 Chapter 5  Spectral functions Laser Detector Channel Mirror Filter Parameter UV (349 nm or 355 nm) A 765 LP 809/82 BP UV809 704 LP 736/64 BP UV736 675 LP 695/40 BP UV695 645 LP 660/30 BP UV660 595 LP 610/30 BP UV610 570 LP 585/30 BP UV585...
  • Page 52 BD FACSymphony™ A5 SE User's Guide Primary Channel Secondary Channel Alexa Fluor™ 488 B510 B537 Alexa Fluor™ 647 R680 R675 Alexa Fluor™ 700 R730 R710 Alexa Fluor™ R720 R730 R710 Alexa Fluor™ YG584 YG602 YG585 R660 R675 APC-Cy7 R780 YG825...
  • Page 53 Chapter 5  Spectral functions Primary Channel Secondary Channel BV711 V710 V750 BV750 V750 V785 BV786 V785 V845 FITC B510 B537 NovaBlue 510 B510 B537 NovaBlue 530 B537 B510 NovaBlue 555 B537 B576 NovaBlue 585 B660 B602 NovaBlue 610_30S B660 B675 NovaBlue 660_40S R675 R680...

  • Page 54: Autofluorescence

    Unstained UV446 UV515 Available from BD as members of the BD Horizon Brilliant™ Blue family of dyes. Available from BD as members of the BD Horizon Brilliant™ Ultraviolet family of dyes. Available from BD as members of the BD Horizon Brilliant™ Violet family of dyes.

  • Page 55: Spectral Unmixing Software Considerations

    Chapter 5  Spectral functions Spectral unmixing software considerations When you perform spectral unmixing in BD FACSDiva™ software, you can also view compensated (non-spectral) data in the software. The data view is selected in the Cytometer window on the Compensation tab, using the View Data dropdown list.
  • Page 56 “conventional” detectors for each fluorochrome in compensation mode, and will also be used for gating to define positive populations in your single-stained controls. Labeling the parameter is necessary for spectral unmixing in BD FACSDiva™. See the tables in Spectral function overview (page 48) for information about the parameters and the primary parameters associated with some commonly used commercial dyes.

  • Page 57: Setting Up A New Spectral Experiment

    2. Create a new specimen by clicking the New Specimen button on the Browser toolbar and rename it. 3. Verify that the base configuration, FACSymphony A5 SE Default Configuration, is selected as described in Verifying the configuration and user preferences (page...
  • Page 58 97). 6. Set the Current Tube Pointer to the previously created sample tube with labeled parameters. 7. Create any plots, gates, and statistics needed for data analysis. For details, refer to the BD FACSDiva™ Software Reference Manual. Note: In addition to the dot and density plots, contour plots, and histogram plots for single-parameter data used in compensation cytometry, the BD FACSymphony™ A5 SE flow cytometer features the ability to...

  • Page 59: Creating A Spectral Unmixing Matrix

    Chapter 5  Spectral functions The detector names displayed along the x-axis are formed by combining the first character of the laser color with the center value of the filter bandpass range for that detector. The plot supports the display of specific populations or combinations of populations, and displays live data during acquisition.
  • Page 60 BD FACSymphony™ A5 SE User's Guide 3. Click OK to create the controls. This adds the controls to the experiment Browser window, under Compensation Controls, and creates a tab for each control on the worksheet. If you assigned AutoF to a detector in the experiment layout, an AutoF control tube will display here too, as shown in the following...
  • Page 61 Chapter 5  Spectral functions h. If necessary, adjust the position and width of the P2 gate to span the positive population on the histogram plot. 6. Select Experiment > Compensation Setup > Calculate Spectral Unmixing. The screen displays the Single Stained Setup dialog. 7.

  • Page 62: Recording And Analyzing Spectral Data

    BD FACSymphony™ A5 SE User's Guide Note: To view the saved settings, select menu item Cytometer > Catalogs and select the Compensation Setup tab. The Type column on this tab classifies settings created using Calculate Spectral Unmixing as Spectral and settings created using Calculate Compensation as Compensated.
  • Page 63 You can elect to switch between spectral unmixed and traditional compensation values, by toggling between Spectral and Compensated in the dropdown menu, to compare the plot data in each case. 4. To export spectral BD FACSDiva™ FCS files: a. Select the file or files to export, right-click and select Export > Unmixed FCS Files.
  • Page 64 Right-click the experiment in the browser and select Export > Experiment. 6. If you want to repeat this experiment later, create an experiment template. For instructions about how to do this, refer to the BD FACSDiva™ Software Reference Manual. 7. Delete experiments in the Browser panel as required.

  • Page 65: Repeating A Spectral Experiment

    For instructions about how to create an experiment template, refer to the BD FACSDiva™ Software Reference Manual. 1. In the Browser, select a location to create the new experiment. If desired, create a new folder (from the menu bar, select Experiment > New Folder) and rename it.
  • Page 66 BD FACSymphony™ A5 SE User's Guide The Experiment Templates dialog is displayed. 3. In the tab containing the experiment template that you want to load, select the template and click OK. 4. Confirm the parameters and labels are correct for the assay that you want to repeat.

  • Page 67: Optimizing Cytometer Settings

    Optimizing cytometer settings This chapter covers the following topics: Cytometer settings workflow (page 68) Verifying the configuration and user preferences (page 69) Running a performance check (page 71) Setting up a compensation experiment (page 74) Creating application settings (page 77) Recording compensation controls (page 78) Calculating compensation (page 81)

  • Page 68: Cytometer Settings Workflow

    Manual compensation Compensation setup automatically calculates compensation settings. If you choose to perform compensation manually, not all of the following instructions apply. For detailed instructions, see the BD FACSDiva™ Software Reference Manual. First-time users If you are performing the procedures in this workflow for the first time, you should be familiar with BD FACSDiva™...

  • Page 69: Verifying The Configuration And User Preferences

    Chapter 6  Optimizing cytometer settings About the examples The examples in this chapter use a 4-color bead sample with the following fluorochromes: FITC PerCP-Cy5.5 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.
  • Page 70 BD FACSymphony™ A5 SE User's Guide 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. This configuration must always be used for spectral cytometery workflows.

  • Page 71: Running A Performance Check

    Chapter 6  Optimizing cytometer settings 5. If the CST Mismatch dialog opens, click Use CST Settings. 6. Select Edit > User Preferences. 7. Click the General tab and select the Load data after recording checkbox. See theBD FACSDiva™ Software Reference Manual for more information about cytometer configurations and user preferences.
  • Page 72 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.
  • Page 73 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.

  • Page 74: Setting Up A Compensation Experiment

    BD FACSymphony™ A5 SE User's Guide Setting up a compensation experiment Introduction This topic describes how to create an experiment in a new folder, specify the parameters of the experiment, and add compensation tubes. Creating an experiment To create an experiment: 1.
  • Page 75 Chapter 6  Optimizing cytometer settings Cytometer settings display in the Inspector. 2. Make sure the parameters you need appear on the Parameters tab in the Inspector. a. Click a Parameter name to display the available fluorochromes in the Parameters list. b.
  • Page 76 BD FACSymphony™ A5 SE User's Guide c. For this example, select FITC from the menu. 3. Delete any unnecessary parameters. Note: Unnecessary parameters may only be deleted when running in compensation mode. a. Click the selection button (to the left of the parameter name) to select the parameter.

  • Page 77: Creating Application Settings

    Chapter 6  Optimizing cytometer settings Creating application settings Introduction This topic describes how to create application settings. About application settings Application settings are associated with a cytometer configuration and include the parameters for the application, area scaling values, PMT voltages, and threshold values, but not compensation. Each time a performance check is run for a configuration, the application settings associated with that configuration are updated to the latest run.

  • Page 78: Recording Compensation Controls

    The application settings are saved to the catalog. Next step Recording compensation controls (page 78) 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.
  • Page 79 Chapter 6  Optimizing cytometer settings Creating compensation tubes To create compensation control tubes: 1. Select Experiment > Compensation Setup > Create Compensation Controls. The Create Compensation Controls dialog opens. 2. Set or clear the Include separate unstained control tube/well and/or Include Generic checkboxes as needed.
  • Page 80 BD FACSymphony™ A5 SE User's Guide 5. Verify that the population of interest is displayed appropriately on the FSC vs SSC plot and adjust voltages if necessary. 6. Adjust the P1 gate to surround only the singlets. 7. Right-click the P1 gate and select Apply to All Compensation Controls.

  • Page 81: Calculating Compensation

    Chapter 6  Optimizing cytometer settings 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.
  • Page 82 BD FACSymphony™ A5 SE User's Guide...

  • Page 83: Recording And Analyzing Data

    Recording and analyzing data This chapter covers the following topics: Data recording and analysis workflow (page 84) Preparing the workspace (page 84) Recording data (page 85) Analyzing data (page 87) Reusing an analysis (page 91)

  • Page 84: Data Recording And Analysis Workflow

    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 67).

  • Page 85: Recording Data

    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.
  • Page 86 BD FACSymphony™ A5 SE User's Guide 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 87: Analyzing Data

    Chapter 7  Recording and analyzing data 11. Before recording, preview the data on the MyData worksheet to verify that all expected populations are visible and the data is similar to the previous sample. 12. Click Record Data. 13. When event recording has completed, remove the second tube from the cytometer. 14.
  • Page 88 BD FACSymphony™ A5 SE User's Guide 7. Select all plots except the FSC vs SSC plot, and use the Plot tab in the Inspector to show only the singlet population. 8. Select all plots, and click the Title tab in the Inspector.
  • Page 89 Chapter 7  Recording and analyzing data 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.
  • Page 90 BD FACSymphony™ A5 SE User's Guide 12. Print the analysis. Your global worksheet analysis objects should look like the following.

  • Page 91: Reusing An Analysis

    Chapter 7  Recording and analyzing data More information Reusing an analysis (page 91) Reusing an analysis Introduction This topic describes how to use 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.
  • Page 92 BD FACSymphony™ A5 SE User's Guide The analysis objects from the MyDataAnalysis global worksheet are copied to the Beads_001_Analysis normal worksheet. Double-click the Beads_001 tube in the Browser to view the analysis. Applying an analysis to normal worksheets You can apply the global worksheet analysis to multiple tubes (on a single normal worksheet) by selecting multiple tubes before pasting the analysis.

  • Page 93: Manual Settings

    Manual settings This chapter covers the following topics: About laser delay (page 94) Optimizing laser delay (page 95) Adjusting area scaling (page 97)

  • Page 94: About Laser Delay

    Yellow-green laser (longest) delay The laser delay setting in BD FACSDiva™ software is used to re-align the signals so they can be measured and displayed on the same time scale, ensuring that the signals from each laser are assigned to the proper event.

  • Page 95: Optimizing Laser Delay

    Chapter 8  Manual settings Optimizing laser delay Introduction This topic describes how to optimize the laser delay using BD FACSDiva™ software. Before you begin To optimize the delay for a given laser, you must acquire events from a sample with a fluorescence signal excited by that laser.
  • Page 96 BD FACSymphony™ A5 SE User's Guide 5. Set an initial laser delay value only for the laser you are optimizing. If you are optimizing the violet laser, set its delay to –35 µs. If you are optimizing the UV laser, set its delay to –65 µs.

  • Page 97: Adjusting Area Scaling

    About area scaling The area of a pulse is calculated by BD FACSDiva™ using measured height and width measurements. It is sometimes important to verify that the area calculation and the height measurement are equivalent by adjusting the factor applied to the area. The required area scaling factor changes based on sheath pressure and particle size.
  • Page 98 BD FACSymphony™ A5 SE User's Guide 3. In the Inspector, click the Parameters tab and select the H checkbox to select the height for each parameter. 4. On the global worksheet, create the following plots and histograms: FSC vs SSC dot plot...
  • Page 99 Chapter 8  Manual settings Your worksheet should look similar to the following figure. 7. Expand the new specimen, then set the current tube pointer to tube_001. 8. Install the FITC-positive control tube onto the loading port and click Load in the Acquisition Dashboard. 9.
  • Page 100 BD FACSymphony™ A5 SE User's Guide 11. Adjust the FSC area scaling. a. Click the Laser Tab in the Cytometer window. b. Adjust the FSC area scaling factor until the FSC-A signal matches the FSC-H signal: Increase the area scaling factor if the FSC-A signal is lower than FSC-H.
  • Page 101 Chapter 8  Manual settings 14. Adjust the red laser area scaling factor until the APC-A signal matches the APC-H signal, if needed.
  • Page 102 BD FACSymphony™ A5 SE User's Guide...

  • Page 103: Troubleshooting

    Troubleshooting This chapter covers the following topics: Cytometer troubleshooting (page 104) Electronics troubleshooting (page 109)

  • Page 104: Cytometer Troubleshooting

    BD FACSymphony™ A5 SE User's Guide Cytometer troubleshooting Introduction This topic describes possible problems and recommended solutions for BD FACSymphony™ A5 SE 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 44).
  • Page 105 Set the PMT voltage higher for the threshold parameter. parameter is set too low 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 27).
  • Page 106 Make sure that the cytometer configuration in the software matches the optical filters in the assignment cytometer and the configuration is as expected. Laser is not functioning Call your BD service representative. No signal in red laser channels (when red laser is installed) Possible causes...
  • Page 107 Increase the threshold level. See the BD FACSDiva™ Software Reference Manual for instructions. PMT voltage for the threshold Set the PMT voltage lower for the threshold parameter. See the BD FACSDiva™ parameter is set too high Software Reference Manual for instructions. Sample is too concentrated Dilute the sample.
  • Page 108 BD FACSymphony™ A5 SE User's Guide Distorted scatter parameters Possible causes Recommended solutions Cytometer settings are improperly Optimize the scatter parameters. See the BD FACSDiva™ Software Reference adjusted Manual for instructions. Air bubble in the sheath filter or Purge the air from the filter. See Removing air bubbles (page 27).

  • Page 109: Electronics Troubleshooting

    1. In BD FACSDiva™ software, select Cytometer > Connect. workstation and 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.
  • Page 110 BD FACSymphony™ A5 SE User's Guide...

  • Page 111: Supplies And Consumables

    Supplies and consumables This chapter covers the following topics: Ordering information (page 112) Beads (page 112) Reagents (page 113) Equipment (page 113)

  • Page 112: Ordering Information

    BD FACSymphony™ A5 SE User's Guide Ordering information To order spare parts and consumables from BD Biosciences: Within the US, call (877) 232-8995. Outside the US, contact your local BD Biosciences customer support representative. Worldwide contact information can be found at bdbiosciences.com. Beads Introduction This topic lists the QC and CS&T beads available.

  • Page 113: Reagents

    ® Detergent Solution BD Biosciences 660585 Concentrate BD FACSClean™ BD Biosciences 340345 Dyes and fluorochromes BD Biosciences, Life Technologies, or Sigma – ® Chlorine bleach Clorox or other major supplier (to ensure that – (5% sodium hypochlorite) the bleach is at the correct concentration and free of particulate matter)
  • Page 114 BD FACSymphony™ A5 SE User's Guide...
  • Page 115 42 conventions BD detergent solution 37 safety symbols 8 BD FACSClean solution 113 creating BD FACSDiva software See software 8 analysis objects 87 BD FACSFlow sheath fluid 113 global worksheets 84 BD FACSFlow solution 27 statistics view 87 BD FACSFlow supply system 24, 38 CS&T particles 112...
  • Page 116 BD FACSymphony™ A5 SE User's Guide starting 24 flow cell troubleshooting 104 draining 31 flow rate control buttons 16 fluid control buttons PRIME 15 DAPI, cleaning after using 37 RUN 15 data STANDBY 15 analyzing 84, 87 fluidics gating 87...
  • Page 117 Index lasers options 20 quality control (QC) performance check 71 particles 112 quality control (QC) particles 112 troubleshooting 109 maintenance recording Bal seal 42 compensation settings 79 cytometer shutdown 38 compensation tubes 78 sample tube O-ring 44 data 84-85 schedule 36 removing air bubbles, filter 28 scheduled 38 replacing...
  • Page 118 BD FACSymphony™ A5 SE User's Guide sheath container components shown 26 user preferences 71 defined 19 depressurize 26 preparing 26 waste air filter sheath filter component shown 30 components shown 41 waste container 19 ordering 113 alarm 19 removing air bubbles 27...
  • Page 120 Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, California 95131 USA BD Biosciences European Customer Support Tel +32.53.720.600 help.biosciences@bd.com bdbiosciences.com ResearchApplications@bd.com...

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