iQue Forecyt® Reference Guide (Version 9.0)

Compensation

About Compensation

All iQue® assay kits have templates with compensation factored in. However, if you are performing other types of experiments, determining the fluorochromes best suited to your experiment is a critical aspect of experiment design. There are a variety of factors to consider because there is a wide brightness range for fluorochromes; some antigens are dimly expressed while others are bright. That’s why matching colors to antibodies in the reagent panel is critical to the success of your experiment.

Your experiment design should factor in signals from one reagent optically interfering with signals from another. This spectral overlap—spillover—occurs when light emitted from one fluorochrome leaks, or spills over, into the channel detecting the fluorescent signal emitted by another fluorochrome. Spillover can be corrected by compensation. Compensation removes the spillover fluorescence from the "wrong" channel and distinguishes dim populations from negative populations. The more careful you are in the design of your experiment the less compensation will be required.

The following diagram illustrates one of the most common spillover situations: FITC into PE.