BD FACSCelesta Flow Cytometer - Page 110

BD FACSCelesta Flow Cytometer User’s Guide110
Using the Compensation tab of the Cytometer window in
BD
FACSDiva software, you can adjust the PE-%FITC spectral
overlap value. Compensation is optimal when the positive and
negative FITC populations have the same medians in the PE
parameter statistics.
The following image shows the FITC spillover optimally
compensated out of the PE parameter.
Once fluorescence compensation has been set for any sample, the
compensation setting remains valid for a subsequent dim or bright
sample (provided the signal is not saturated), because
compensation subtracts a percentage of the fluorescence intensity.
The following figure illustrates this principle. Although the signals
differ in intensity, the percentage of the FITC spillover into the PE
detector remains constant.
PE
FITC
FITC-positive
population
Unstained
particles
PE
FITC
Unstained
particles
FITC-positive
population
Matching medians