Compensation controls...

Are a correction factor applied to each channel in use on a flow cytometer to correct for spectral spillover of unintended fluorophores into their neighboring channels.  Compensation controls aid in accurate gating strategies and the avoidance of artefactual events.

Compensation controls are typically necessary for any flow experiment. Fluorophores emit along a spectrum around the peak emission. As a bandpass filter will “catch” wavelengths within its range indiscriminately, compensation should be applied in order to correct for spectral overlap between fluorophores. Compensation controls are single color-stained controls. They can be prepared as stained cells, however, compensation beads are the most robust reagent to use for this purpose.

It should be noted that high compensation values are not inherently indicative of poor reagents. These values can be directly influenced by voltage changes. What’s more important is that the compensation applied is accurate so that the cell populations can be more easily visualized and gated upon.

The four standard practices listed below help to ensure an accurate calculation of compensation:

  1. The reagents applied to the compensation controls, and experimental samples must be from the same vial of reagent and ideally treated the same until acquired.
  2. The autofluorescence of the compensation control must be identical for both the negative and positive events.
  3. Each compensation control's positive population must be at least as bright as any positive signal in the experimental sample.
  4. To ensure precise compensation, collect enough events to estimate median fluorescence in all channels as well as the negative population.