There are a few basic principles to remember when designing compensation controls for an experiment. Since compensation controls are critical to the determination of what we call positive or negative for a given marker, they are absolutely critical to the success of the instrument. The definition of a compensation control is simple: for each fluorophore used in the experiment, a single-stained cell or bead sample must also be prepared.
The important rules to remember are:
Fortunately, compensation is easily accomplished by software when the correct controls are used. The software will calculate spillover values and apply them to the data, and the data will be properly compensated (Figure 12).
Figure. 12. Fluoresence compensation corrects for spectral overlap. FITC single-stained cells showing fluorophore being detected in PE channel before (A) but not after compensation (B).
Chapter 3: Gates and Regions
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