FACS And Compensation
SP, May 2005
As illustrated in the bottom diagram on this page, compensation may be required when using multiple fluorophores whose emission signals overlap. Compensation is usually done for pairs of adjacent channels. E.g., assuming that 10% of FITC signal (FL1 channel, green) spills into the FL2 channel (orange) being used for PE-signal, the true PE-signal is judged by subtracting 10% equivalent of the FL1 signal from the FL2 signal.
Compensation settings are usually adjusted during data acquisition, though compensation can also be performed during data analysis with certain software such as FlowJo.
In some cases, e.g., when using propidium iodide staining for cell viability (dead cells stain; FL3 channel, but leaks into FL2) and a PE-tagged antibody (FL2 channel), compensation may not be needed if you just want to exclude PI-positive cells, being dead, from analysis.
Notes
— Proper compensation requires 'compensation control' samples. These are cells stained for only one fluorophore. Say, in an experiment both PE and FITC fluorophores are used - i.e., all test samples are stained with both. The compensation controls to set up would be a tube with only the PE fluorophore and another one with only the FITC fluorophore. Proper compensation would be be achieved when the median PE (and FITC) fluorescence of the sample using both FITC and PE fluorophores is same as that of the control sample using just the PE (or FITC) fluorophore.
— When altering compensation settings, adjust with respect to the median of the populations (and not lower or upper boundaries)
— Each of the different test samples in an experiment, as long as they all have same autofluorescence (e.g., all samples are peripheral blood monocytes), does not need its own compensation control. However, it is best to choose the 'likely-brightest' sample for preparing the compensation control.
— If a sample is too bright that keeping it within scale (visible within the display window) leads to invisibility of the negative sample (median falling below axis), one would need to use a different compensation control sample. Such a sample, instead of being a non-stained sample, could be a dimly-stained sample prepared by using, say the primary antibody, at a hundred-more-fold dilution.
— It is best to set up such controls everytime an experiment is done instead of relying on old settings.
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