BD FACSymphony A1 Flow, User Manual

Page: 63 / 126

Before you begin
Perform the cytometer setup procedure and run a performance check for the configuration that will be used for
the application.
Procedure
To create application settings:
1. In the open experiment, right-click Cytometer Settings in the Browser , then select Application Settings >
Create Worksheet.
A second global worksheet is added with the plots created according to the selections in the Parameters tab.
2. Load the unstained control tube onto the cytometer.
3. In the Cytometer window, optimize the PMT voltages for the application.
l
Optimize the FSC and SSC voltages to place the population of interest on scale.
l
Optimize the FSC threshold value to eliminate debris without interfering with the population of interest.
l
If needed, increase the fluorescence PMT voltages to place the negative population appropriately for
your sample type.
4. Unload the unstained control tube from the cytometer.
5. Load the multicolor sample onto the cytometer or load single-color control tubes and verify each
fluorochrome signal separately.
6. Verify that the positive populations are on scale.
If a positive population is off scale, lower the PMT voltage for that parameter until the positive population
can be seen entirely on scale.
7. Unload the multicolor sample.
8. Place a tube containing less than 1 mL DI water on the SIP and put the cytometer on standby.
9. (Optional) Save the application settings by right-clicking Cytometer settings in the Browser , then selecting
Application Settings > Save.
10. In the Save Application Settings dialog, enter a descriptive name for the application settings.
Chapter 5 Optimizing cytometer settings 63