Chapter 5: Optimizing cytometer settings 79
Use the gray boxes and crosshairs to guide your optimization.
2. Load the unstained control tube onto the cytometer.
3. In the Cy
tometer window, optimize the PMT voltages for the
application.
• Optimize the FSC and SSC voltages to place the population
of
interest on scale.
• Optimize the FSC threshold value to eliminate debris
without interfering with the population of interest.
• If needed, increase the fluorescence PMT voltages to place
the negative population within the gray boxes. Align the
center of the negative population with the crosshair visible
in the gray box.
Note: Do not decre
ase the fluorescence PMT voltages. Doing
so can make it difficult to resolve dim populations from the
negative population.
4. Unload the unstained control tu
be from the cytometer.
5. Load the multicolor sample onto the cytometer or load single-
co
lor control tubes and verify each fluorochrome signal
separately.
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