BD FACSCanto II User manual

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BD FACSCanto II Flow Cytometer
Quick Reference Guide
This guide contains instructions for both BD FACSCanto™ clinical and
BD FACSDiva™ software. Follow only those sections that apply to the
software you are running.
Starting Up
1Turn on the cytometer main power.
2Start up the computer, launch the software, and
log in.
3Make sure the software is connected to the cytometer; if needed,
connect manually (choose Cytometer or Instrument > Connect).
4Check fluid levels in the Status or Instrument window.
Empty the waste or replace fluid containers, if needed.
5If automatic cleaning is disabled, run the fluidics startup procedure.
6Check the flow cell for air bubbles; if needed, run the de-gas flow cell
procedure.
7Check that laser warmup has finished.
To prevent fluid overflow, make sure there is no tube on the sample
injection tube (SIT) during fluidics startup.
or
BD FACSCanto clinical software
BD FACSDiva software
BD FACSDiva software
BD FACSCanto clinical software

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Daily Setup: BD FACSCanto Clinical Software
Set up the cytometer with BD FACSCanto clinical software, even if you will
optimize and run samples using BD FACSDiva software.
1Prepare BD FACS™ 7-color setup beads.
2Run the cytometer setup procedure for the bead lot you prepared.
Follow the Cytometer Setup Wizard to complete setup. When
prompted, install the tube containing the setup beads, or the rack
containing the beads tube and optimization samples.
3If you are running samples manually, unload the tube when prompted.
•Hold the tube while moving the aspirator arm to the left.
•Remove the tube from the SIT.
•Release the aspirator arm and wait for cleaning to finish.
4(Optional) When setup is complete, view and print the Setup Report.
5Click to optimize the settings, or click .
6Review the Levey-Jennings report for trends in the setup data.
Optimizing Instrument Settings: BD FACSCanto Clinical Software
Before you record data for a sample, optimize the detector voltages,
spectral overlap, and threshold settings for the sample type and
fluorochrome to be run.
1Follow the prompts on the screen to proceed with optimization.
•Click Yes to save results.
•Choose the panel type and the X and Y parameters, and click
.
•If you are using the Loader for optimization, click .
•Click at the Cytometer Setup Optimization screen.
•If you are running tubes manually, install the first optimization tube
when prompted, and click .
•Click .

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2Optimize settings, if needed.
•To adjust the optimization gate, click , make the
needed adjustments, and then click .
•To adjust detectors, thresholds, or spectral overlap settings, click on
the corresponding tab and use controls to adjust the settings.
3If there are more tubes for that panel type, click , and then
.
Otherwise, click , and then view and print the Setup
Report for optimized settings.
4Click , remove the tube (if prompted), and then click .
Optimizing Instrument Settings: BD FACSDiva Software
Before you record data for a sample, optimize the PMT voltages, compensation,
and threshold settings for the sample type and fluorochrome.
1Verify the instrument configuration.
2Create an experiment.
3Select the experiment-level settings, and change, add, or delete
parameters as needed.
4Apply setup results.
•Right-click the experiment-level settings, and choose Apply Setup.
•Select a setup, and click Apply.
5Create compensation controls; click OK to remove the setup link.
•If needed, edit labels for the Compensation Controls.
6Optimize instrument settings using an unstained control sample.
•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.
•Set the fluorescence histograms to show grids for each log decade.
•Optimize the voltages to place the negative population for each
fluorescent parameter within its first log decade.

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7Click ; remove the unstained control tube when
recording is finished.
8Record data for each single-stained control.
9Adjust the gates around the positive populations on the histogram for
each stained control tube.
10 Calculate compensation, and name the resulting setup.
Running Samples: BD FACSCanto Clinical Software
1Prepare a worklist.
2Install the first sample tube (manual acquisition) or the Loader rack
(automated acquisition), and click .
3When asked if you want to save the worklist, click No.
4(Loader option only) Click to run tubes manually.
5(Optional) To optimize settings during initial acquisition, click ,
and then .
Adjust settings, and click to resume.
6For manual acquisition, remove the sample tube when prompted and
install the next tube; repeat until data for all tubes has been recorded.
7Inspect the lab report for each sample on the worklist.
Double-click the Status field to see a sample’s report.
Running Samples: BD FACSDiva Software
1Create a new specimen and tubes; rename them appropriately.
2Create a global worksheet and rename it.
3Use Experiment Layout to define tube labels and enter events to record.
4On the global worksheet, create appropriate plots.
5Install the first tube onto the cytometer; acquire data for the
corresponding tube in the Browser.
6While data is being acquired, draw a gate around the population of
interest; set the other plots to show data from this population.
7Click ; remove the tube when recording is finished.
8Install the next tube.
9Repeat until data has been recorded for all tubes.

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Shutting Down: BD FACSCanto Clinical Software
1Create a worklist with a two-tube panel; enter Clean as the Sample ID.
2Click ; when asked to save the worklist, click No.
3(Loader option only) Click to run tubes manually.
4When prompted, install a tube containing ≤3 mL of BD™ FACSClean
solution or 10% bleach.
5Click . Wait 5 minutes, and then click .
6When prompted, unload the tube.
7Install a tube with ≤3 mL of DI water; repeat steps 5 and 6.
8Choose File > Exit, and select Run fluidics shutdown and exit.
9When fluidics shutdown completes, turn off the power.
Shutting Down: BD FACSDiva Software
1Install a tube with ≤3 mL of BD FACSClean solution or 10% bleach,
and click .
2After 5 minutes, click and remove the tube.
3Repeat steps 1 and 2 with DI water.
4Run fluidics shutdown.
5Turn off the instrument main power.
To prevent fluid overflow, do not leave a tube on the SIT during fluidics
shutdown.
To prevent fluid overflow, do not leave a tube on the SIT during fluidics
shutdown.

BD, the BD logo, BD FACS, BD FACSDiva, and BD FACSCanto are trademarks of Becton, Dickinson and Company.
http://bdbiosciences.com/
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BD FACSCanto Clinical Software Toolbars
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