Why perform compensation?
Compensation is the process of subtracting the spectral spillover from one fluorchrome into the detector of another.
Emission spectra of fluorochrome overlap
An example of spectral overlap can be observed between FITC and PE. Fluorescence caused by FITC during a single stain procedure must be subtracted or compensated from the PE detector.
How is compensation performed?
- Setting PMT voltages.
- Detecting autofluorescence for each fluorochrome in the panel.
- Select single stain controls for each flurochrome in the panel.
What are single stain controls?
- Compensation beads
While cells may seem like a good choice, beads are a great alternative as the cell source may be limited or the antigen of interest is expressed at low levels.
Compensation points to consider
- Always prepare a tube of unstained cells for PMT volage set up.
- Do not use negative beads to set up voltages.