BD FACSCanto Flow Cytometer
Quick Reference Guide
BD FACSCanto Flow Cytometer
Quick Reference Guide
Main
Page
Starting Up
2
Optimizing Instrument Settings
3
Running Samples
4
Running Samples from an existing experiment
5
Shutting Down
6
Registration
6
Maintaining the Database
7
Exporting an experiment
7
Exporting files
7
Importing an experiment
7
BD FACSDiva Sofware Toolbars
8
Blockage Prevention
9
Blockage Removal
9
Personal Protective Equipment (PPE) Requirements
10
Handling of Infectious Samples
10
Hazardous Waste
10
Troubleshooting
11
Contact Details
11
1
BD FACSCanto Flow Cytometer
Quick Reference Guide
Starting Up
1 Turn on the cytometer main power (the green botton on the left of the
cytometer)
2 Start up the computer (Ask password to the facility manager)
3 Launch the BD FACSDiva™ software, Choose your session and log in
4 Open Instrument Window
. Make sure the software is connected to the
cytometer; if needed, connect manually (choose Instrument > Connect).
5 Run the fluidics startup procedure.
To prevent fluid overflow, make sure there is no tube on the sample injection tube (SIT)
during fluidics startup.
7 Check that laser warmup has finished.
2
Optimizing Instrument Settings
Before you record data for a sample, optimize the PMT voltages, compensation,
and threshold settings for the sample type and fluorochrome.
1 Create an experiment or duplicate an existing one without dada.
2 Select the experiment-level settings, and change, add, or delete parameters
as needed (in the inspector).
3 Choose Instrument > Instrument Setup > Create Compensation Controls
• If needed, edit labels for the Compensation Controls.
4 Optimize instrument settings using an unstained control sample.
• Open the Adquisition Dashboard.
• Place the current tube pointer next to the unstained control tube,
install the tube, and click
• Adjust FSC and SSC voltages, and the FSC threshold, if needed.
• On the Unstained Control worksheet, adjust the P1 gate, and apply
gate changes to the remaining compensation controls.
• Optimize the voltages to place the negative population for each
fluorescent parameter within its first log decade.
5 Click Record; remove the unstained control tube when recording is finished
To Remove Tube: click Remove Tube button in the Adquisition Control Frame, a
progress dialog appears. Hold your sample tube with one hand while you push the
aspirador arm all the way to the left with the other hand. Remove the tube from the
sample injection tube (SIT). Release the aspirador arm. When the progress dialog box
disappears, you can load the next tube onto the SIT.
6 Record dada for each single-stained control
7 Create gates around the positive population on the histogram for each
stained control tubes
8 Calculate compensation
• Choose Instrument > Instrument Setup > Calculate Compensation
• Name the compensation Setup, and click OK
3
Running Samples
1 Create a new specimen and tubes; rename them appropriately.
2 Create a global worksheet and rename it.
3 Use Experiment Layout to define tube labels and enter events to record.
4 On the global worksheet, create appropriate plots.
5 Install the first tube onto the cytometer; acquire data for the
corresponding tube in the Browser.
6 While data is being acquired, draw a gate around the population of
interest and set the other plots to show data from this population.
7 Click
; remove the tube when recording is finished.
8 Install the next tube.
9 Repeat until data has been recorded for all tubes.
Running samples from an existing experiment
4
1 Duplicate an experiment withouth data (Open an old experiment, click right button
and choose duplicate without data).
Before you record data for a sample, optimize the PMT voltages,
compensation, and threshold settings for the sample type and fluorochrome.
2 Optimize instrument settings using an unstained control sample.
• Open the Adquisition Dashboard.
• Place the current tube pointer next to the unstained control tube,
install the tube, and click
• Open the Instrument Window
. In parameters you can adjust the
voltages. When you had adjusted all the parameters click record and
when the cytometer arribes to the marked number of events it will stop
automatically. You can stop the adquisition manually, click stop
recording and stop adquiring: the sample is recorded likewise.
3 Remove the unstained control tube when recording is finished
To Remove Tube: click Remove Tube button in the Adquisition Control Frame, a
progress dialog appears. Hold your sample tube with one hand while you push the
aspirador arm all the way to the left with the other hand. Remove the tube from the
sample injection tube (SIT). Release the aspirador arm. When the progress dialog box
disappears, you can load the next tube onto the SIT.
4 Install the next tube and click record.
If you have more than a color adquire the compensation controls. Adjust compensation
manually or proced to automatical compensation (page …). If you adjust compensation
mannually you have to paste the new compensation to the global instrument settings (paste
espectral overlap)
5 Repeat until data has been recorded for all tubes.
5
Shutting Down
1 Install a tube with ≤3 mL 10% bleach, and click adquire data.
2 After 5 minutes, click
and remove the tube.
3 Repeat steps 1 and 2 with DI water.
4 Run fluidics shutdown.
To prevent fluid overflow, do not leave a tube on the SIT during fluidics
shutdown.
5 Turn off the instrument main power.
Registration
Register the time you use the cytometer in the notebook Registre d’utilització that is
beside the cytometer. Anotate the cleaning maintenance performed and the
incidences that may occur.
6
Maintaining the Database
To maintain an optimal size of the database it is important that always
export and delete the experiments from de sofware and backup them in
another location (usuaris folder)
Every 6 months the Cytometry Service will back up usuaris folder and will delete
all the items. Its convenient that you always save your experiments in your own
device.
Exporting an Experiment
1 Select one or more Experiment icons in the browser.
2 Choose File > Export > Export Experiment
3 Make appropiate selections in the Export Experiment dialog box
Select Delete experiments after export checkbox. To avoid confusion, store Exported
Experiments in a folder separate from Expoted Files.
Exporting Files
1 Select Experiment, Specimens, or tubes to export
2 Choose File > Export > Export FCS
Alternatively right-clik the selected item and choose Export FCS.
3 Specify the FCS version and parameterss to export in the dialog box. Click
OK
To make an exported file compatible wint BD CellQuest Pro Sofware, export it as FCS
2.0
4 In the Save export dialog box, verify the file storage location
Importing an experiment
1 Select the folder where you want to locate the imported experiment
2 Choose File > Import > Experiment
3 Select the folder containing the required Experiment and click Import.
7
8
Blockage Prevention
Proper sample preparation will reduce the chance of blockages during your
acquisition. It is recommended to routinely:
1 Filter samples through 70 micron nylon mesh
2 Add 1-5 mM EDTA to the samples prior to acquisition.
3 Add 0.1% DNAse to your sample prior to acquisition if your cell viability is low
(<70%).
Blockage Removal
1 In instrument, Cleaning modes, select Degas flow cell. Repeat 3 times.
1 Run 10% bleach at HI flow rate for 5 minutes.
2 Run FACSRinse at HI flow rate for 5 minutes.
4 Run MilliQ water at HI flow rate for 5 minutes.
If the cytometer is still blocked, seek help from the facility manager.
9
Personal Protective Equipment (PPE) Requirements
Laboratory gowns and gloves must always be worn when acquiring samples
on the flow cytometers. Place a protective cover on the computer keyboard and
mouse when adquiring samples
Handling of Infectious Samples
This protocol applies to ALL human samples, virus-infected samples of any
species and alive microorganisms.
1 If it is possible, fix cells in 1-2% PFA
2 Wear gloves, and laboratory gown
3 Ensure that there is 100 ml neat bleach in the waste tank.
4 Place the plastic cover on the computer keyboard and mouse.
5 Post-acquisition of files, follow the standard shutdown procedure.
6 Clean bench area and cytometer with cleaning solution
7 Let the waste sit for at least 1 hour.
8 Empty the waste tank into the sink and flush away the contents with copious
volumes of water.
You can ask the facility manager to do that for you
Hazardous Waste
Dispose Biohazardous waste in the biohazard waste container.
Dispose Cytostatic waste (propidium Iodide, formaldheid…) in the cytostatic
container.
10
Troubleshooting
A comprehensive troubleshooting guide is located in the FACSCanto Reference
Manual and FACSDiva Sofware Reference Manual. If the problem persists,
then consult the facility manager.
Contact Details
Manuela Costa
Tècnic Superior de Suport a la Recerca

Laboratori d’Immunogia.
Institut de Biotecnologia i Biomedicina
Universitat Autònoma de Barcelona
09193 Bellaterra. Barcelona

x 8948

[email protected]

661303611
x 8996
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