BD FACSCanto User Manual

High throughput sampler

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Page 1 BD High Throughput Sampler User’s Guide for the BD LSR II BD FACSCanto BD FACSCanto II bdbiosciences.com Part No. 642224 Rev. A bdbiosciences.com April 2007 Part No. 642224 Rev. A June 2007 BD Biosciences Asia Pacific Brazil Canada BD Biosciences...
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 BD Biosciences, it is the buyer/user’s responsibility to install and maintain up-to-date virus protection software. BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation. BD Biosciences is not liable for any claims related to or resulting from buyer/user's failure to install and maintain virus protection.
Page 5: Table Of Contents
Sample Processing ..........Chapter 2: BD FACSDiva Software Overview Workspace Components .
Page 6 BD FACSCanto II ........
Page 7 Acquisition Troubleshooting ........BD FACSDiva Troubleshooting ........
Page 8 Packing the Unit for Shipping ........Placing the HTS into Its Shipping Container ..... . Index viii BD High Throughput Sampler User’s Guide...
Page 9: About This Guide
About This Guide This guide describes how to set up and operate the BD™ High Throughput Sampler (HTS) option with the BD™ LSR II, BD FACSCanto™, and BD FACSCanto™ II flow cytometers. You should know how to operate your flow cytometer before using the HTS option. For important safety information, refer to the safety booklet provided with the HTS option.
Page 10: Conventions
Print” means to choose Print from the File menu. Ctrl-X When used with key names, a dash means to press two keys simultaneously. For example, Ctrl-P means to hold down the Control key while pressing the letter p. BD High Throughput Sampler User’s Guide...
Page 11: Technical Assistance
Customers outside the US and Canada, contact your local BD representative or distributor. If you need to send your HTS unit back to BD Biosciences for repair, see Appendix B on page 157 for instructions. Note that depot repair procedures might be different outside of the United States.
Page 12 BD High Throughput Sampler User’s Guide...
Page 13: Chapter 1: Introduction
Introduction The BD High Throughput Sampler (HTS) is a compact, high-speed sample loading device for use with the BD LSR II, BD FACSCanto, and BD FACSCanto II flow cytometers. BD FACSDiva software controls the sample loader, providing automated acquisition of samples from a multiwell plate.
Page 14: Hts Hardware Overview
Table 1-1 Default throughput mode settings Standard High-Throughput Setting Mode Mode Sample Flow Rate (µL/sec) Sample Volume (µL) Mixing Volume (µL) Mixing Speed (µL/sec) Number of Mixes (cycles) Wash Volume (µL) Approximate Acquisition Time (min) BD High Throughput Sampler User’s Guide...
Page 15 For more information, see Loader Settings on page 38. The HTS unit is installed on the cytometer by a BD Biosciences service engineer. Once installed, the HTS enables quick conversion of the flow cytometer from tube- to plate-based acquisition. Figure 1-1 HTS installed on a BD LSR II flow cytometer...
Page 16: Components
Plate holder—moves left to right and front to back to position plate so the probe can pick up sample Primary pump and valve—enables mixing and aspiration of sample; delivers sample to flow cell in standard mode Power switch and LED indicator for HTS unit BD High Throughput Sampler User’s Guide...
Page 17 Figure 1-3 HTS unit for BD LSR II and BD FACSCanto—rear view Probe assembly—moves front to back and up and down to transfer sample between plate holder and injection port/wash station Fluidics tubing: sheath (clear) and waste (orange) Sheath filter—filters incoming sheath fluid to HTS unit Injection port/wash station—provides interface for sample injection...
Page 18 Figure 1-4 HTS unit for BD FACSCanto II—rear view Probe assembly—moves front to back and up and down to transfer sample between plate holder and injection port/wash station Fluidics tubing: sheath (clear, with in-line filter) and waste (orange) Door sensor cable connector—cable detects if safety door is open Injection port/wash station—provides interface for sample injection...
Page 19: Cytometer Connections
Sheath and waste travel between the cytometer and the HTS unit via connectors in the cytometer interface panel (see Figure 1-5, Figure 1-6, and Figure 1-7). The BD LSR II interface panel also includes an acquisition mode switch, which controls pressure at the cytometer sample injection tube (SIT).
Page 20 Figure 1-5 Cytometer interface panel [BD LSR II] Tube/Plate acquisition mode switch sheath connector waste connector waste tubing sheath tubing cytometer power cable support bracket communication cable base plate HTS positioning screw Figure 1-6 Cytometer interface panel [BD FACSCanto] communication...
Page 21: Sample Coupler
SIT protector. The SIT protector is a modified sleeve that prevents the sample injection tube from bending during installation of the HTS sample coupler. For the BD FACSCanto and BD FACSCanto II, the sample coupler is installed on the SIT, while the aspirator arm rests against the sample coupler. Flip the aspirator arm bar to the back (as shown in Figure 1-8 on page 22) to ensure it does not come in contact with the HTS probe.
Page 22 Figure 1-8 Cytometer SIT during plate-based acquisition – BD LSR II (left); BD FACSCanto (right) and BD FACSCanto II (bottom) SIT protector sample sample coupler coupler aspirator aspirator arm bar SIT protector sample aspirator aspirator arm bar • For instructions on switching the cytometer from tube to plate acquisition, see Setting Up for Plate-Based Acquisition on page 59.
Page 23: Sample Processing
NOTICE [BD LSR II] Soft standby mode is not available when you are acquiring samples in Plate mode. Refer to your BD LSR II User’s Guide for information about soft standby mode. Sample Processing During acquisition, the HTS processes samples differently based on the throughput mode.
Page 24 BD High Throughput Sampler User’s Guide...
Page 25 Understanding Volumes There are different volumes to take into consideration when choosing loader settings. Figure 1-9 shows different well volumes and Table 1-3 defines each volume type. Figure 1-9 Well volumes sample probe available volume total volume plate-dependent dead volume well Table 1-3 Volume Type Volume Type...
Page 26 (10 µL or less), increase the sample rate (1 µL/sec or greater), minimize the number of mixes (2 or less), and decrease the wash volume (400 µL or less). NOTICE The BD FACSCanto II maximum event rate is 10,000 events/second. BD High Throughput Sampler User’s Guide...
Page 27: Chapter 2: Bd Facsdiva Software Overview
BD FACSDiva Software Overview This chapter describes the BD FACSDiva software features necessary to operate the BD High Throughput Sampler with the BD LSR II, BD FACSCanto, and BD FACSCanto II flow cytometers. For an in-depth description of software components not described in this chapter, refer to the BD FACSDiva Software Reference Manual.
Page 28: Workspace Components
Windows containing the main application components are displayed within the workspace. Display additional windows by clicking buttons in the Workspace toolbar. Figure 2-1 BD FACSDiva workspace displaying an open experiment and the Plate window BD High Throughput Sampler User’s Guide...
Page 29 The type you choose becomes the default. Browser toolbar 96-well U-bottom plate 96-well V-bottom plate 96-well flat-bottom plate open 384-well flat-bottom plate experiment current tube pointer closed experiment Chapter 2: BD FACSDiva Software Overview...
Page 30 To show or hide the Plate Controls, Acquisition Setup, or Acquisition Status sections of the Acquisition Dashboard, right-click the Acquisition Dashboard in any blank area (except for Basic controls) to display the shortcut menu. You can resize the Acquisition Dashboard using standard Windows methods. BD High Throughput Sampler User’s Guide...
Page 31 Settings in the Plate window (see Loader Settings on page 38). Set up, acquire, and analyze plate-based experiments in the Plate window. Use this window also to select the throughput mode and adjust loader settings. Chapter 2: BD FACSDiva Software Overview...
Page 32: Plate Window
Figure 2-2 Setup view of Plate window plate toolbar plate legend/filter view tabs throughput mode loader status specimen list HTS settings plate layout See the following sections for a description of components in, and the functionality of, each view. BD High Throughput Sampler User’s Guide...
Page 33: Setup View Components
Well settings – When cytometer settings are added to a well, the cytometer settings icon appears in the lower-left corner of the well. Chapter 2: BD FACSDiva Software Overview...
Page 34 For more information on well status, see Plate Layout on this page and Table 2-2 on page 36. BD High Throughput Sampler User’s Guide...
Page 35 Table 2-1 Assessing well status during setup or acquisition Well Status selected for acquisition selected for acquisition and contains previously recorded data Chapter 2: BD FACSDiva Software Overview...
Page 36 If you export a data file or experiment that contains incomplete data, the software cannot recognize that the file is incomplete. BD High Throughput Sampler User’s Guide...
Page 37 Compensation Controls You can create label-specific compensation controls. Refer to the BD FACSDiva Software Reference Manual for information. Throughput Mode The default mode at startup is high throughput. Select throughput mode by clicking the corresponding mode button in the Setup view. You can also select throughput mode in the Plate Inspector.
Page 38 You cannot change HTS settings during acquisition or when a sequence is in process. NOTICE If multiple wells, with different loader settings, are selected, a red highlight appears around the loader settings that are different. BD High Throughput Sampler User’s Guide...
Page 39 During acquisition in standard mode, BD HTS aspirates the selected sample volume (2–200 µL) plus an additional 20 µL from the well. During acquisition in high throughput mode, BD HTS aspirates a fixed 22 µL per well even though you can select a sample volume between 2–10 µL.
Page 40 Wash Volume (µL) 200–800 200–800 a. BD recommends a mixing volume that is one-half the available volume. See Understanding Volumes on page 25 and Mixing on page 26. NOTICE A mixing volume that is larger than the available volume introduces air bubbles into the sample.
Page 41 • link to a setup • apply compensation controls • apply and save application settings • create an experiment layout For additional information about these commands, refer to the BD FACSDiva Software Reference Manual. Chapter 2: BD FACSDiva Software Overview...
Page 42 To paste to a different area of the plate and make a new specimen, select an empty well. From the shortcut menu, choose Paste > Well(s). BD High Throughput Sampler User’s Guide...
Page 43: Setup View Functions
Use the Setup view to perform the following functions: • Define loader settings • Optimize cytometer settings while running setup controls • Monitor well status during acquisition • Acquire and record wells manually or automatically • Analyze plate data Chapter 2: BD FACSDiva Software Overview...
Page 44 Assigning Keywords You can create and assign keywords at the experiment or well level. For information on creating experiment keywords, refer to the BD FACSDiva Software Reference Manual. For instructions on creating and assigning keywords at the well level, see Assigning Keywords on page 77.
Page 45 To copy settings from one well to another, click to select the well, then right-click it and choose Copy Cytometer Settings. Select the well you wish to copy to, then right-click it and choose Paste Cytometer Settings. Chapter 2: BD FACSDiva Software Overview...
Page 46 The system flushes any remaining sample that was aspirated. • Run Well(s)/Stop Well(s) toggle button—starts recording for all wells selected in the sequence; terminates the sequence after recording data for the current well. The system flushes any remaining sample that was aspirated. BD High Throughput Sampler User’s Guide...
Page 47 Dashboard. You can acquire, record, and restart acquisition on a single selected well as long as a sequence is not currently running. For detailed information on the Basic acquisition controls, refer to the BD FACSDiva Software Reference Manual. To start manual mode, do one of the following: •...
Page 48: Analysis View Components
• reviewing and analyzing data collected from a plate • starting a batch analysis For information on performing these functions, see Analysis View Functions on page 51. BD High Throughput Sampler User’s Guide...
Page 49 If a well has no value assigned, the well is white. To revert back to the default Analysis view, click the keyword in the Keywords list. Figure 2-3 Plate layout showing Keyword Analysis view Add Keyword button Keyword list Chapter 2: BD FACSDiva Software Overview...
Page 50 The Keywords list contains the selected keywords for all samples and wells on the plate (Figure 2-3). A maximum of 15 keywords can be added to the list. Add keywords to the list using the Add Keyword button. See Add Keyword Button for more information. BD High Throughput Sampler User’s Guide...
Page 51: Analysis View Functions
Shortcut Menu Right-click a selected well at the Analysis view to open the shortcut menu. The following commands are available in the shortcut menu. Analysis View Functions Perform the following functions in the Analysis view. Chapter 2: BD FACSDiva Software Overview...
Page 52 For example, Figure 2-6 displays values for the keyword Concentration. In this example, four different concentrations were assigned to wells on the plate. The Keyword Analysis view shows the concentration value assigned to each well. Figure 2-6 Keyword Analysis view BD High Throughput Sampler User’s Guide...
Page 53 Analyzing Plate Data Analyze plate data the same way you would analyze tube data. For detailed information, refer to the BD FACSDiva Software Reference Manual. Performing Batch Analysis Use the batch analysis feature to automatically advance a selected set of data for multiple wells through an analysis template on a global worksheet.
Page 54 BD High Throughput Sampler User’s Guide...
Page 55: Chapter 3: Running Samples
Chapter 2. If you are running the system for the first time, BD Biosciences recommends that you practice running a sample plate using BD Calibrite™ beads or a similar control sample. The following topics are covered in this chapter: •...
Page 56: Starting Up
If necessary, perform a sheath fluid exchange to use the appropriate sheath solution. See Exchanging the Sheath Fluid on a BD LSR II on page 96 or Exchanging the Sheath Fluid on a BD FACSCanto and BD FACSCanto II on page 98.
Page 57 Double-click the shortcut on the desktop. Verify that no other software applications are running before you start acquisition. BD FACSDiva software performance time can be severely affected if multiple applications are running at the same time. At the Log In dialog, choose your user name, enter your password, and click OK.
Page 58 Figure 3-1 Cytometer window connectivity status [BD FACSCanto and BD FACSCanto II] Perform a fluidics startup. Choose Cytometer > Fluidics Startup. The following message appears. Click OK to begin fluidics startup. When fluidics startup is complete, the following message appears.
Page 59: Setting Up For Plate-Based Acquisition
[BD LSR II] Place the cytometer in Run mode, then choose HTS > Prime to prime the HTS unit. When the priming is complete, click OK in the dialog. NOTICE To prime the cytometer, press the Prime button on the fluid control panel on the cytometer itself.
Page 60 HTS probe. This position also ensures that the aspirator arm is able to detect an installed sample coupler. Figure 3-2 Installing the sample coupler on the BD FACSCanto or BD FACSCanto II sample coupler aspirator arm (moved towards back) ‘...
Page 61 If necessary, install the HTS unit. For instructions, see Installing the HTS Unit on page 170. Switch the acquisition control switch to plate mode ( Remove the tube of DI water from the SIT. Remove the DCM sleeve. Unscrew the tube retainer that holds the DCM sleeve onto the SIT and carefully remove the sleeve.
Page 62 Note that there should be a gap between the tightening nut and the bottom of the SIT protector (Figure 3-4 on page 63). If you don’t see a gap, unscrew the tube retainer, push the SIT protector all the way up, and retighten the tube retainer. BD High Throughput Sampler User’s Guide...
Page 63 Figure 3-4 Installing the sample coupler on the BD LSR II SIT protector tightening nut sample coupler NOTICE Make sure the sample coupler is securely connected to the SIT. Turn on the HTS. The power switch is on the right side of the HTS unit. See Figure 3-5.
Page 64: Creating Experiments
Creating Experiments An experiment is a group of elements used to acquire and analyze data from the BD High Throughput Sampler. The Browser is where you create experiments and access stored data. Here are two ways to add experiments to the Browser.
Page 65 Figure 3-6 User Preferences dialog NOTICE Show file identifier (GUID) in statistics view ensures that the GUID keyword—the FCS file’s unique identification number—appears in the header of statistics views. Tube-specific worksheet and Save analysis after recording NOTICE through global worksheet options do not apply to plate runs on the HTS. Click the New Folder button ( ) in the Browser toolbar.
Page 66 NOTICE The HTS is compatible only with standard-depth 96- or 384-well plates. BD plates can be ordered from BD Discovery Labware. For ordering information, visit the BD Discovery Labware website at bdbiosciences.com/discovery_labware/. Table 3-1 Plates compatible with the BD High Throughput Sampler Plate Type Well Capacity (µL)
Page 67: Setting Up The Plate
Make sure you choose the plate type that corresponds to the plate you will be using. BD FACSDiva software cannot verify that the chosen plate matches the plate on the HTS unit. If you choose the wrong plate, the probe could hit the plate between wells or strike the bottom of a well, resulting in damage to the cytometer.
Page 68 Plate Inspector unless you acquire individual wells manually. See Running Wells Manually on page 47 for more information. In the Browser, click on the Experiment (global) Cytometer Settings. Delete unnecessary parameters in the Parameters tab of the Cytometer Inspector. BD High Throughput Sampler User’s Guide...
Page 69 The parameters should match those shown in the following figure. Click and drag to select wells A1 and A2 in the plate layout. Click the Add Setup Controls button ( ) in the Plate toolbar to add setup control wells to the experiment. These two wells will be used for unstained control to set threshold and PMTs.
Page 70 Click and drag to select the following wells, and then click the Add Specimen Wells button ( ) to add two specimens to the plate layout. Wells Name C1–C12 Specimen_001 D1–D12 Specimen_002 You can change the specimen name using the Specimen Inspector. BD High Throughput Sampler User’s Guide...
Page 71 The plate layout should look similar to Figure 3-9. Figure 3-9 Setup view Choose Experiment > Experiment Layout, and click the Labels tab (Figure 3-10 on page 72). Define fluorophore labels for both samples. Fluorophore Label Fluorophore Label FITC CD14 PE-Cy7 CD19 CD16+CD56...
Page 72 There are other fast ways to create labels. Refer to the BD FACSDiva Software Reference Manual. The Experiment Layout should look similar to Figure 3-10. Figure 3-10 Labeled fluorophores in Experiment Layout Click the Acquisition tab in the Experiment Layout (Figure 3-11), and set the number of Events to Record.
Page 73 See the BD FACSDiva Software Reference Manual for more ways to add events. Figure 3-11 Acquisition tab of Experiment Layout Click OK to save the changes.
Page 74: Setting Up The Worksheet
Create the following dot plots choosing the appropriate parameters. • FITC vs PE • PerCP-Cy5.5 vs PE-Cy7 • APC vs APC-Cy7 Show a population hierarchy. The worksheet should look similar to Figure 3-12 on page 75. BD High Throughput Sampler User’s Guide...
Page 75 Figure 3-12 Plots on global worksheet Chapter 3: Running Samples...
Page 76: Exporting A Plate As A Template
Template in the shortcut menu. The Export Plate Template Wizard appears. The bold text at the top of the dialog tells you what to do at each screen. See Figure 3-13 on page 77. BD High Throughput Sampler User’s Guide...
Page 77: Assigning Keywords
Click Next and then Finish to view the remaining screens. Assigning Keywords Keywords are used to enter information about the wells, such as sample type and preparation details. For more information, refer to the BD FACSDiva Software Reference Manual. Chapter 3: Running Samples...
Page 78: Preparing For Acquisition
Make sure the plate corresponds to the type selected in the software. Orient the plate with well A1 at the back-right corner of the stage. See Figure 3-14 on page 79. BD High Throughput Sampler User’s Guide...
Page 79 Replace the HTS safety cover. [BD LSR II] Press the RUN button on the cytometer. Do not put the BD LSR II in Standby during HTS acquisition. BD FACSDiva software cannot continue the run when the cytometer is in Standby, and it can cause damage to the flow cell.
Page 80 NOTICE Acquisition stopping time is determined by BD FACSDiva software, according to sample volume/sample rate. BD recommends you set the target number of events high enough so that you do not run out of events before you reach the software-based acquisition stopping time. For more information on stopping time, see Running Samples Automatically in Sequence on page 46 and Running Wells Manually on page 47.
Page 81: Performing Compensation
Performing Compensation Once you have optimized cytometer settings, you are ready to run your compensation controls and calculate compensation. NOTICE You can run compensation controls only once during an experiment. If you need to re-run compensation controls, you must create a new experiment. Select wells B1 through B7 in the plate layout.
Page 82: Acquiring Data
Name the compensation setup after the experiment, and click OK. For information on performing compensation, refer to the BD FACSDiva Software Reference Manual. Compensation controls can be run individually through their normal worksheets using Basic Controls in the Acquisition Dashboard.
Page 83: Pausing The Bd High Throughput Sampler
(See Plate Layout on page 35.) At the end of each day or shift, clean and service the cytometer as described in Daily Maintenance on page 104. Pausing the BD High Throughput Sampler To pause during a run, click Pause in the Acquisition Dashboard.
Page 84: Stopping The Bd High Throughput Sampler
Click Stop Plate or Stop Well(s) in the Acquisition Dashboard. The BD High Throughput Sampler finishes the sequence in progress, stops, and then the following message appears: NOTICE If you are using the Basic Controls to acquire, simply click Stop Acquiring Click OK to close the dialog.
Page 85 Choose File > Export > Experiment Template. In the shortcut menu, select Export > Experiment Template. Make sure you choose the Experiment Template command, and not the Experiments command. The Export Experiment Template Wizard appears. The bold text at the top of the dialog tells you what to do at each screen.
Page 86: Analyzing Data
Analyzing Data This section shows you how to use Analysis view and the batch analysis feature in BD FACSDiva software to analyze data from plate-based acquisition. Displaying Keywords Click the Analysis tab to display the Analysis view. The Analysis view shows the acquisition status of each well.
Page 87 Figure 3-15 Add Keywords dialog Alt-Shift-Click the keywords Concentration, Lymphocytes, and Granulocytes from the Available Keywords list (on the left side of the dialog). The Concentration, Lymphocytes, and Granulocytes keywords were created for this example. Click to move the keywords to the Selected Keywords column; click OK.
Page 88 Choose the appropriate print options, and click OK. Save the printout for your records. Click the keyword Lymphocytes in the Keyword list. The Keyword view displays the values for the keyword Lymphocytes. See Figure 3-17 on page 89. BD High Throughput Sampler User’s Guide...
Page 89 Figure 3-17 Keyword Analysis view showing Lymphocytes values Print the Lymphocytes Keyword view and legend. Click the keyword Granulocytes in the Keyword list. The wells that were designated with the keyword Granulocytes are colored yellow-green, but do not list a value since none was assigned. Chapter 3: Running Samples...
Page 90: Performing Data Analysis
Print the Granulocytes Keyword view and legend. Performing Data Analysis Analyze plate data in the same manner you would analyze tube data using BD FACSDiva software. For detailed instructions, refer to the BD FACSDiva Software Reference Manual. Performing Batch Analysis Do the following to set up batch analysis.
Page 91 Click the Analysis tab to display the Analysis view. In the global worksheet, verify that gates are adjusted to enclose the appropriate populations. In the plate layout, select a well or wells for batch analysis, right-click to open the shortcut menu, and then choose Batch Analysis. NOTICE If you wish to print, you must assign a printer before starting batch analysis.
Page 92 A new header row is added if you add or delete statistics or parameters during batch analysis. You cannot add, remove, or edit statistics views while the batch is running. BD High Throughput Sampler User’s Guide...
Page 93: Maintaining Data
All data is processed using scales from the tube where the current tube pointer is set. For information on biexponential scales, refer to the BD FACSDiva Software Reference Guide. Preferred global worksheets can be used with wells and specimens. For information on using preferred global worksheets, refer to the BD FACSDiva Software Reference Manual.
Page 94: Returning To Tube-Based Acquisition
Returning to Tube-Based Acquisition Returning to Tube-Based Acquisition on the BD LSR II To return to tube mode, you need to remove the sample coupler and the SIT protector, reinstall the DCM sleeve, place the acquisition control switch in tube mode, and remove the HTS as described in this section.
Page 95 Placing HTS Unit into Long-Term Storage on page 132 To prevent personal injury or damage to the sampler, disconnect the BD High Throughput Sampler from the cytometer and remove the unit before running tube-based acquisition. Chapter 3: Running Samples...
Page 96 If you exchanged BD FACSFlow with BD FACS sheath solution with surfactant, install the HTS sample coupler, then prime the system twice by choosing HTS > Prime. This will exchange the fluid in the syringes with BD FACS sheath solution with surfactant.
Page 97: Returning To Tube-Based Acquisition On The Bd Facscanto
To avoid bending the SIT, pull straight down on the coupler. Do not pull the coupler at an angle. NOTICE For the BD FACSCanto, place the sample coupler in the clamp on the catch tray when the coupler is not in use (see Figure B-11 on page 173). Chapter 3: Running Samples...
Page 98 Exchanging the Sheath Fluid on a BD FACSCanto and BD FACSCanto II Perform this procedure to exchange one sheath fluid with another. For example, when switching from plate-based to tube-based acquisition, follow these steps to exchange BD FACS sheath solution with surfactant with BD FACSFlow.
Page 99 A dialog appears informing you that the sheath exchange is complete. Click OK. NOTICE When the sheath type is BD FACSFlow, the sheath fluidics level indicator ( ) will be green. When the sheath type is FACS sheath solution with surfactant, the level indicator will be white.
Page 100: Shutting Down
If you exchanged BD FACSFlow with BD FACS sheath solution with surfactant, install the HTS sample coupler, then prime the HTS twice by choosing HTS > Prime. This will exchange the fluid in the syringes with BD FACS sheath solution with surfactant.
Page 101: Quitting The Software
A dialog appears when fluidics shutdown is complete. Click OK. Quitting the Software If you have a BD FACSCanto and BD FACSCanto II, you must perform a fluidics shutdown before quitting the software (see Fluidics Shutdown on page 100). Choose File > Quit.
Page 102 BD High Throughput Sampler User’s Guide...
Page 103: Chapter 4: Maintenance
Maintenance This chapter provides maintenance procedures to keep your HTS in good working order. The following maintenance procedures are covered in this chapter. • Daily Maintenance on page 104 • Monthly Maintenance on page 111 • Periodic Maintenance on page 114 •...
Page 104: Daily Maintenance
Perform the cleaning procedure at the end of every day or shift while the cytometer is in plate-based mode. Allow 15 minutes to complete this procedure. Materials Needed • BD FACSClean or a fresh 10% bleach solution (1 part bleach in 9 parts DI water) • BD FACSRinse solution •...
Page 105 Running Daily Cleaning Choose HTS > Clean. The Plate Templates dialog appears (Figure 4-1 on page 105). Figure 4-1 Plate Templates dialog Select the Daily Clean - 96 well U-bottom template, if not already selected. If you do not have a U-bottom plate for cleaning, you can set up your own cleaning template.
Page 106 Do not click OK at this time. Fill the wells of a 96-well plate according to the following table. Wells Solution Volume (PL) A1–A4 BD FACSClean B1–B4 DI water a. or a 10% bleach solution BD High Throughput Sampler User’s Guide...
Page 107 To ensure that the 10% bleach solution retains its full germicidal effect, prepare a fresh solution daily. NOTICE If you wish to run BD FACSRinse solution as part of the daily cleaning procedure, repeat the daily cleaning using BD FACSRinse solution in wells A1– A4 and DI water in wells B1–B4.
Page 108 The cytometer goes through a homing sequence, and cleaning begins. Note that the cleaning procedure can take up to 15 minutes. Click OK when the completion message appears. Remove and discard the multiwell plate, or rinse it for use on another day. BD High Throughput Sampler User’s Guide...
Page 109: Cytometer Inspection And Servicing
Sample coupler Check for leaks around the sample coupler on the cytometer SIT. If necessary, tighten the top nut to secure the sample coupler to the SIT. BD FACSCanto/BD FACSCanto II BD LSR II tightening nut If the coupler continues to leak after you tighten the fitting, remove and then reinstall the coupler as follows.
Page 110 See Replacing a Pump Syringe on page 125. • [BD LSR II and BD FACSCanto] Absorbent pad Inspect the absorbent pad at the back of the HTS unit to make sure it is not saturated. Replace the pad, if needed.
Page 111: Monthly Maintenance
DI water through the lines for 30 minutes. Allow 60 minutes to complete the monthly cleaning procedure. To perform the monthly cleaning you will need DI water and BD FACSClean or a fresh 10% bleach solution (1 part bleach in 9 parts DI water).
Page 112 A dialog appears while cleaning is in progress; this can take up to 30 minutes. Remove the tank and replace the cleaning solution with DI water. Reinstall the tank. A progress dialog appears while rinsing is in progress; this can take up to 30 minutes. BD High Throughput Sampler User’s Guide...
Page 113 Click OK when monthly cleaning is complete. NOTICE If you have been running BD FACSRinse solution as part of a biweekly cleaning procedure, you can repeat the monthly cleaning using BD FACSRinse solution followed by DI water. Disconnect the tank, fill it with sheath solution, and reconnect it.
Page 114: Surface Inspection And Cleaning
Use universal precautions when handling cytometer surfaces. Wear suitable protective clothing and gloves. • Use BD FACSClean (or a 10% bleach solution) followed by DI water to wipe down the HTS enclosure and cover on a monthly basis, or when needed.
Page 115 Component Part No. Replacement Schedule Procedure Sampler coupler and 339340 yearly or when needed page 123 tubing (BD FACSCanto and BD FACSCanto II) Syringes 339047 yearly or when needed page 125 Probe 34389017 yearly or when needed page 129 Quick-connector O-ring...
Page 116: Replacing The Sample Injection Tubing
Perform the monthly cleaning procedure to decontaminate the sample injection port/wash station. See Monthly Cleaning on page 111. Turn off the HTS power switch (BD LSR II and BD FACSCanto), or turn off the cytometer (BD FACSCanto II). For easy access, remove the HTS unit.
Page 117 Remove the sample injection tubing (A) from the HTS. • If necessary, disconnect the top hex screw (B) from the secondary pump using the 5/16-in wrench in the accessory kit (Figure 4-5 on page 116 and Figure 4-6). Figure 4-6 Hex screw on secondary pump secondary pump valve tubing (F) hex screw (B)
Page 118 The magnetic force will secure the sample injection port/wash station in place. Reconnect the secondary pump valve tubing (F) to the secondary pump by tightening the hex screw (B). Reinstall the HTS unit. Follow the instructions for Installing the HTS Unit on page 170. BD High Throughput Sampler User’s Guide...
Page 119: Replacing The Hts Sheath Filter
Loosen the positioning screw holding the HTS to the cytometer support bracket (Figure 4-8). Figure 4-8 Loosening the positioning screw (example shown for BD LSR II) support bracket positioning screw Slide the HTS unit towards you by doing the following: •...
Page 120 Figure 4-9 Sheath line and filter (example shown for BD LSR II) sheath line filter [BD FACSCanto] Remove the catch tray. Pull the HTS towards you slightly to allow room for removing the catch tray. Label the sheath line connectors.
Page 121: Cleaning The Air Filter
Reinstall the HTS unit. [BD FACSCanto] Install the rear catch tray by routing the sample coupler underneath the catch tray tongue and sample coupler slot. Push the tray in, making sure the coupler line is not crimped or kinked.
Page 122 Remove the filter retainer. The filter retainer is located within the air filter element in the base plate of the unit. Example of filter for BD LSR II and BD FACSCanto filter retainer •...
Page 123: Replacing The Sample Coupler And Tubing
Remove the filter and clean or replace it as needed. filter filter retainer To clean the filter, rinse it well with water. Dry it completely before you reinstall it. Place the clean filter inside the filter retainer. Reinstall the filter retainer. Center it over the fan guard and push evenly on the opposite edges to snap it into place.
Page 124 1/6 to 1/4 turn. If leaking is observed, tighten the fitting no more than an additional 1/8 turn. Reinstall the HTS unit on the cytometer. For detailed instructions, see Installing the HTS Unit on page 170. BD High Throughput Sampler User’s Guide...
Page 125: Replacing A Pump Syringe
Remove the liquid from the syringe by performing steps 2 through 11 in Placing HTS Unit into Long-Term Storage on page 132. Turn off the HTS power switch (BD LSR II and BD FACSCanto), or turn off the cytometer (BD FACSCanto II). Keep the software running on the workstation.
Page 126 Push the syringe plunger up to the top of the syringe barrel. Loosen the plunger lock screw approximately three full turns counterclockwise (Figure 4-12 on page 127). Lower the plunger drive by pushing down on the plunger lock screw. See Figure 4-12 on page 127. BD High Throughput Sampler User’s Guide...
Page 127 Figure 4-12 Lowering the plunger valve plunger drive plunger lock screw Unscrew the syringe from the valve. Grasp the metal ring at the top of the syringe and turn counterclockwise. Discard the syringe into a biohazardous sharps container. Install the new syringe (Figure 4-13 on page 128). •...
Page 128 [BD LSR II and BD FACSCanto] Turn on the HTS. • [BD LSR II] Choose HTS > Prime to prime the fluidics until the plunger barrel is full of sheath fluid. If you see any bubbles, continue priming until the bubbles are gone.
Page 129: Replacing The Probe
Replacing the Probe The probe is located at the end of the probe assembly arm. Replace the probe whenever it is bent or permanently clogged. A spare probe is included in the accessory kit. All cytometer surfaces and hardware that come in contact with biological specimens can transmit potentially fatal disease.
Page 130: Replacing Quick-Connector O-Rings
Install a new O-ring, pushing it into place with your thumbs. Reconnect the quick-connector. Replacing SIT Protector and O-Ring (BD LSR II Only) The SIT protector is a modified sleeve that prevents the sample injection tube from bending during installation of the HTS sample coupler. An O-ring between the SIT protector and the tube retainer keeps the protector in place.
Page 131 Remove the safety cover from the HTS. Detach the sample coupler from the cytometer SIT. Remove the SIT protector (Figure 4-16). Unscrew the tube retainer and slide the SIT protector straight down. To avoid bending the SIT, do not slide the SIT protector at an angle. Figure 4-16 Removing the SIT protector tube retainer SIT protector...
Page 132: Placing Hts Unit Into Long-Term Storage
Use universal precautions when handling cytometer components. Wear suitable protective clothing, eyewear, and gloves. Placing the HTS for BD LSR II in Long-Term Storage Remove the HTS safety cover. Locate and loosen the positioning screw that secures the HTS to the cytometer support bracket (Figure 4-17 on page 133).
Page 133 Figure 4-17 Loosening the positioning screw support bracket positioning screw Grasp the sides of the HTS unit and slide it forward until it meets the stop in the cytometer support bracket. Lift the HTS unit off of the cytometer support bracket by doing the following: To prevent personal injury or damage to the HTS unit, do not slide the HTS unit out so far that it becomes unbalanced or the strain of the tubing bends...
Page 134 Put the end of the purging assembly line into a 500-mL beaker containing DI water. Sheath (B) connector in port purging assembly line beaker of DI water Put the safety cover on the HTS. BD High Throughput Sampler User’s Guide...
Page 135 Choose HTS > Prime; repeat nine times. Priming will replace the sheath fluid with DI water. Remove the end of the purging assembly line from the DI water and lay it on the benchtop. Remove the purging assembly line and reconnect the HTS tubing to the Sheath (B) port.
Page 136: Unscheduled Maintenance
Placing the HTS for BD FACSCanto or BD FACSCanto II in Long- Term Storage Ensure the sample coupler is installed and the HTS is turned on. Perform a fluidic shutdown. See Fluidics Shutdown on page 100 for details. If required, remove the HTS from the cytometer. See Removing the HTS Unit on page 159.
Page 137 [BD LSR II] Place the cytometer in Run mode, and ensure that the sample coupler is installed. The software automatically primes the HTS pumps during a homing operation. To prevent pressure from building in the flow cell, the cytometer must be in Run mode before the homing sequence begins. If you try to run the Home command when the cytometer is not in Run mode, a software message will remind you that the fluidic system is not ready.
Page 138: Priming The Hts
You might need to perform a motion test when you set up the HTS unit for the first time (ie, after a depot repair procedure), when axis movement is suspect, or when you are directed to do so by a BD Biosciences service representative during cytometer troubleshooting.
Page 139 Note that this test will execute the same sequence whether a 96- or 384-well plate is selected. For the most accurate troubleshooting, use a 384-well plate to verify the mechanism reaches the extreme positions. Choose HTS > Motion and Position Test. The Motion and Position Test dialog appears.
Page 140: Verifying The Sample Probe Position
To run this test, you must select a plate type in BD FACSDiva software and install the corresponding plate on the plate holder. The test can be run with a 96- or 384-well plate.
Page 141 Verify that the sample probe properly performs the following sequence: • The probe travels to the home position. • The probe moves up, and then moves to well A1. The following message appears. Remove or open the safety cover and verify that the probe is in the center of the nearest well (position A1).
Page 142: Reinitializing The Hts
• Extension tubing to the secondary pump valve—There is a male thumbscrew fitting at the sample injection tubing end. BD High Throughput Sampler User’s Guide...
Page 143: Inspecting Hexagonal Fittings
Declogging the SIT Perform this procedure to dislodge a clog in the SIT only when instructed to do so by BD Biosciences. All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease. Use universal precautions when handling cytometer components.
Page 144 Gently pull the cleaning stylus most of the way out of the SIT and then push it back in; repeat several times. Remove the cleaning stylus from the SIT, rinse the stylus with BD FACSClean followed by DI water, and dry the stylus. Replace the sample coupler. Choose HTS > Prime.
Page 145: Chapter 5: Troubleshooting
BD High Throughput Sampler. For additional troubleshooting assistance, refer to the appropriate cytometer user’s guide. If additional assistance is required, contact your local BD Biosciences service representative. See Technical Assistance on page xi. Troubleshooting suggestions in this chapter are grouped under the following headings: •...
Page 146: Hts Troubleshooting
• Replace wash station assembly Sheath float switch time Air in the fluid lines • Choose Cytometer > Cleaning out error during sheath Modes > Prime after Tank Refill. exchange • Bleed air out of sheath filter. BD High Throughput Sampler User’s Guide...
Page 147: Acquisition Troubleshooting
Waste tank back pressure • Ensure waste fittings are connected and not damaged. • [BD LSR II] Ensure waste lines are not kinked. • Ensure antifoam concentrate is added to waste tank. Chapter 5: Troubleshooting...
Page 148 Bubbles in syringe Prime unit. Choose HTS > Prime. No filter cap [BD FACSCanto and BD FACSCanto II] Replace filter cap. Unexpectedly low Insufficient stopping Make sure to set the target number of number of events criteria...
Page 149: Bd Facsdiva Troubleshooting
Well(s) not selected Select a well(s). [BD FACSCanto and Sample coupler not installed Ensure sample coupler is installed BD FACSCanto II only] before cleaning modes are HTS not included in selected. cleaning modes No power Ensure HTS is powered on.
Page 150 BD High Throughput Sampler User’s Guide...
Page 151: Appendix A: Consumables And Replacement Parts
To order spare parts and consumables from within the US, call (877) 232- 8995 or go to bdbiosciences.com. In other countries, contact your local BD Biosciences representative. This information is correct at the time of publication. For up-to-date information, refer to our website (bdbiosciences.com).
Page 152: Cytometer Supplies
34389017 HTS sheath filter (2) 335710 Quick-disconnect O-ring 343618 O-ring sleeve retainer [BD LSR II] 343620 Air filter 336218 Absorbent pad [BD LSR II and BD FACSCanto] 343502 Injection tubing to secondary pump 335451 BD High Throughput Sampler User’s Guide...
Page 153 Anti-foam concentrate, 25 g bottle 334887 Cleaning stylus 343648 Purging assembly line [BD LSR II] 340088 HTS safety cover [BD LSR II and BD FACSCanto] 640404 2-ft serial cable [BD FACSCanto] 640737 HTS catch tray [BD FACSCanto] 640576 HTS power/communication cable [BD FACSCanto II]...
Page 154: Maintenance Log
Injection Tubing Replacing the HTS page 119 Sheath Filter Cleaning the Air Filter page 121 Replacing the Sample page 123 Coupler and Tubing Replacing a Pump Syringe page 125 Replacing the Probe page 129 BD High Throughput Sampler User’s Guide...
Page 155 Monthly Maintenance Procedure Month Completed (Initials/Date) Replacing Quick- page 130 Connector O-Rings Replacing SIT Protector page 130 and O-Ring (BD LSR II Only) Placing HTS Unit into page 132 Long-Term Storage Appendix A: Consumables and Replacement Parts...
Page 156 BD High Throughput Sampler User’s Guide...
Page 157: Appendix B: Depot Repair Procedures
Appendix B Depot Repair Procedures This appendix contains procedures to perform if your BD High Throughput Sampler (HTS) needs repair and depot repair service is available in your region. For a summary of how the depot repair process works, see Depot Repair Overview on page 158.
Page 158: Depot Repair Overview
Depot Repair Overview BD High Throughput Sampler User’s Guide...
Page 159: Removing The Hts Unit
Maintenance on page 111. If you cannot run the system, decontaminate it after removal as described in Cleaning the HTS Unit on page 162. [BD LSR II and BD FACSCanto] Switch off the HTS power. Shut down the cytometer. Remove or open the HTS safety cover.
Page 160 Wear suitable protective clothing and gloves. [BD FACSCanto] Remove the catch tray. [BD LSR II and BD FACSCanto] Lift the HTS unit off of the base plate by doing the following: To prevent personal injury or damage to the HTS unit, do not slide the HTS unit out so far that it becomes unbalanced.
Page 161 [BD FACSCanto II] Lift the front feet of the HTS unit just over the front edge of the enclosure and tilt the unit at a 45° angle towards you to allow you access to the door sensor cable on the right side of the HTS. Unplug the door sensor cable by pulling back on the ferrule.
Page 162: Cleaning The Hts Unit
Figure B-2 Drip reservoir containing absorbent pad [BD LSR II and BD FACSCanto only] absorbent pad Place the sample tubing and attached sample coupler inside the drip reservoir. Cleaning the HTS Unit You must decontaminate both the internal fluidics and external surfaces of your HTS unit before it is repaired.
Page 163: Decontaminating The Fluidics Manually
To prevent shock, verify that the cytometer is turned off and the power cable is detached before you start cleaning. Use a 10% bleach solution to decontaminate the internal fluidics and external surfaces of the HTS unit. To ensure that the 10% bleach solution retains its full germicidal effect, prepare a fresh solution daily.
Page 164 Using a disposable pipette, fill the injection port/wash station completely, being careful not to overfill. Connect the line declogger tool to the waste connector on the back of the unit. Figure B-4 Attaching the declogger tool BD High Throughput Sampler User’s Guide...
Page 165 Slowly pull back the plunger of the declogger tool until all of the bleach solution is drawn into the barrel. Disconnect the declogger tool and discard the bleach solution according to local regulations. Repeat step 2. Reattach the declogger tool, and slowly remove about half of the bleach solution;...
Page 166 (C) thumb screw (D) sample injection tubing (E) connector (G) thumb screw (F) • Unscrew the thumb screw (F) from the connector (G) and remove the secondary pump valve tubing (A) (Figure B-6). BD High Throughput Sampler User’s Guide...
Page 167: Decontaminating External Surfaces
• Discard the secondary pump valve tubing (A) as biohazardous waste. All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease. Treat the secondary pump valve tubing as biohazardous waste and dispose of it according to local regulations. Detach the sample coupler tubing from the secondary pump valve and discard the tubing.
Page 168 Make sure all surfaces are clean. Dispose of the paper towels used for cleaning as biohazardous waste. Rinse all surfaces by repeating steps 1 through 5, substituting DI water for the bleach solution. Allow the cytometer to dry thoroughly. BD High Throughput Sampler User’s Guide...
Page 169: Unpacking The Replacement Unit
Unpacking the Replacement Unit A HTS unit without a cover weighs approximately 22 lb. To prevent personal injury or damage to the cytometer, follow the guidelines on page 169 to remove the replacement HTS unit from its shipping container. NOTICE If you are sending back an existing HTS for depot repair, save all shipping materials and ship the existing unit in this new container.
Page 170: Unpacking The Hts Unit
Any cytometer surface that comes in contact with biological specimens can transmit potentially fatal disease. Use universal precautions when handling cytometer hardware. Wear suitable protective clothing and gloves. To avoid damaging the HTS probe: BD High Throughput Sampler User’s Guide...
Page 171 [BD LSR II and BD FACSCanto] Place the HTS on the base plate/carrier. [BD FACSCanto II] Bring the unit close to the cytometer while you sit in a chair with the unit in your lap. Make sure the unit support legs fit into the bracket grooves. With the legs in the groove, slide the unit as far forward as possible.
Page 172 (see Figure 1-4 on page 18). Connect the sheath line (with filter) and waste lines to their respective ports on the back of the unit (or back of the cytometer for BD FACSCanto II) (see Figure B-10). NOTICE The white connector on the BD FACSCanto II is not used for the HTS.
Page 173 Push the tray in, making sure the coupler line is not crimped or kinked. The clamp on the tray is used for the sample coupler when it is not in use. Figure B-11 Catch tray installed at the back of the BD FACSCanto clamp for sample...
Page 174 If necessary, install the probe (refer to Replacing the Probe on page 129). Connect the sample coupler to the SIT. [BD LSR II] With the SIT protector in place, slide the sample coupler onto the SIT until you reach a hard stop. Make sure the sample coupler tubing is not kinked or twisted.
Page 175: Packing The Unit For Shipping
Make sure the unit was decontaminated according to the procedures in Cleaning the HTS Unit on page 162. Disconnect the sheath and waste lines from the HTS. [BD FACSCanto II] Disconnect the interface/communication cable from the HTS. Initial and date the decontamination labels; stick one on the plate holder and the other on the outside of the shipping carton.
Page 176 Place the top piece of foam over the cytometer. Lay the Cytometer Return form on top of the foam, and close and seal the flaps of the shipping container. BD High Throughput Sampler User’s Guide...
Page 177: Index
110 data 82–83 removing 135 defaults 37, 40 batch analysis 90, 92 high-throughput mode 23, 37 BD High Throughput Sampler See HTS. manual 47 Browser, shown 29 modes 23 optimizing settings for 78 plate-based See plate-based carryover, minimizing 26 acquisition.
Page 178 165 deleting wells and settings 40 shutdown, BD FACSCanto 100 depot repair See repair procedures. startup, BD FACSCanto 58 door sensor cable, BD FACSCanto II 18 tubing, shown 17, 18 fluorophore labels 37 BD High Throughput Sampler User’s Guide...
Page 179 instrument settings copying and pasting 45 GUID 65, 93 interface panel, cytometer 19–21 hardware Keyword Analysis view feature 52, 86 basic functionality 14 keywords components 16 assigning 44, 77 installation 15 viewing assignments 52, 86 part replacement 114 plate sizes 14 hazard symbols x hexagonal fittings, inspecting 143 labels 72...
Page 180 18 inspection and service 110 shown 17, 18, 79 icons 35 layout view 35 legend 33 mode 19, 60 rearranging samples on 44 template 76 toolbar 34 Plate Interface, shown 31 BD High Throughput Sampler User’s Guide...
Page 181 110 tube-based acquisition 95 replacing syringes 125 unpacking replacement unit 169 secondary, shown 17, 18 sample coupler 21–23 BD FACS Canto II 18 BD LSR II 17 quitting software 101 inspection and service 109 sample volume 39 samples...
Page 182 42 replacing 116, 123 speed, mixing 39 thumbscrew fittings 142 standard mode 23, 37 status indicators, well colors 35 stopping HTS 84 unscheduled maintenance 136–144 software 101 user preferences 65 syringes, replacing 125 BD High Throughput Sampler User’s Guide...
Page 183 windows Acquisition Dashboard 30 views Browser 29 Acquisition Dashboard 30 Inspector 31 Analysis 48 Plate 31 Browser 29 worksheet Inspector 31 global 65 plate layout 35 normal 76 Plate window 31 setting up 74 Setup 33 volume minimum sample 39 mixing 39 sample 39 volumes...
Page 184 BD High Throughput Sampler User’s Guide...

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Facscanto ii Lsr ii

BD FACSCanto User Manual

Chapter 1: Introduction

Chapter 2: BD Facsdiva Software Overview

Chapter 3: Running Samples

Chapter 4: Maintenance

Chapter 5: Troubleshooting

Appendix A: Consumables and Replacement Parts

Appendix B: Depot Repair Procedures

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Troubleshooting

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