Compensation Beads...

Are stained with single color reagents that present the machine with enough spectral information from the fluorophore in use to generate an accurate compensation matrix.   

Compensation Controls can be generated using single color stained cells or beads. There are limitations to utilizing cells as compensation controls. They must have enough expression of the antigen to generate enough data points for an accurate calculation of compensation for that fluorophore. There also needs to be enough cells in the sample to distribute amongst all of these different kinds of controls and still have enough for the experiment itself.

The alternative is to use compensation beads. Depending on the vendor, the beads will bind a variety of host isotypes and can be quickly mixed with the antibody of choice to create a compensation control. Single color stained cells are ideal for setting the voltages to be applied to the experiment to ensure the positive and negative populations are both within the ideal linear range of detection. Compensation beads are more accurate to populate enough data from the negative and positive populations for the compensation algorithm to calculate. 

 

 

Either compensation beads or single color controls can be suitable for use as compensation controls. The important factors are that there are enough positive and negative events as demonstrated in the histogram using beads. However, since the PMT voltages applied in the assay are determined based on the biological sample to be analyzed, they may not be optimal for very brightly stained compensation beads. In the histogram where beads are employed, the positive peak is still able to be gated on accurately, but is in danger of going beyond the detectable range. In the second example, when using single color stained cells expressing CD44, the cells in this instance will not be suitable since there are not enough negative cells. In addition, the large variance of the MFI+ population can add to additional error in compensation calculation.