Flow cytometry antibodies, kits, reagents

Chapter 2: Principles of Fluorescence

Compensation Controls

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:

  1. The staining of the compensation control must be as bright as or brighter than the sample. Antibody capture beads can be substituted for cells and one fluorophore conjugated antibody for another, as long as the fluorescence measured is brighter for the control. The exceptions to this are tandem dyes, which cannot be substituted.

    Note: Although it would seem safe to assume that all tandem dyes created with the same donor and acceptor would have the same emission, this is not the case. Tandem dyes from different vendors or different batches must be treated like separate dyes, and a separate single-stained control should be used for each because the amount of spillover may be different for each of these dyes.
  2. The compensation algorithm needs to be performed with a positive population and a negative population (Figure 10). Whether each individual compensation control contains beads, the cells used in the experiment, or even different cells, the control itself must contain particles with the same level of autofluorescence. The entire set of compensation controls may include individual samples of either beads or cells, but the individual samples must have the same carrier particles for the fluorochromes.
  3. The compensation control must use the same fluorophore as the sample. For example, both GFP and FITC emit mostly green photons, but have vastly different emission spectra. You thus cannot use one of them for the sample and the other for the compensation control.
  4. Enough events must be collected for the software to make a statistically significant determination of spillover. Ideally about 5,000 events for both the positive and negative population is ideal. 

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).

Fluoresence compensation corrects for spectral overlap

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).