Ideally, the positive and negative populations in the control samples should be the same type of cell.The background fluorescence (autofluorescence) of the positive population should be the same as the negative control. The positive population should form at least 10% of the total sample population.The positive population should be at least as bright or brighter than any sample the compensation will be applied to.The fluorophore used as a compensation control must match the fluorophore used in the experiment.These controls should only be used to set compensation.The following guidelines should be applied: Compensation is correctly set when the median of the negative population is equal to the median of the positive population in the spillover channelĬompensation controls are required for each fluorophore and should contain a positive and negative population.Avoid fluorophore combinations where a bright fluorophore can spill over significantly into a channel if attempting to detect a dimmer population.Do not forget to check the compensation in all channels
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