It is imperative to NOT include autofluorescence in the compensation calculation, so if the positive and negative have the same autofluorescence, then the autofluorescence contribution to the compensation spillover calculation will be zero. This is because compensation is a subtraction algorithm. However, the positive and negative carrier of a parameter must have the same autofluorescence. The error in the compensation coefficient is the sum of the absolute errors in the MFI's of both the negative and the positive control the latter has an inherently much larger absolute error than the former.Īny carrier for binding fluorochromes can be used for single stain compensation controls, such as cells or particles. The bottom line is that because the compensation coefficients are computed based on the RATIO of the DIFFERENCE in MFI's (of the spillover channel and the primary channel), so small absolute errors in the position of the negative control become irrelevant as the positive controls become brighter. Compensation is only about estimating the slope. At a MFI of 100,000, the spillover becomes apparent and then compensation value can be accurately assessed. For example, if a spillover is so low that a MFI of 10,000 doesn't cause enough spillover to be above autofluorescence, then the system assumes no compensation is necessary. Only brightest matters, and that is so that low spillovers can be accurately estimated. In addition, there are three rules for “good” compensation controls:ġ) Controls need to be at least as bright or brighter than any sample to which the compensation will be appliedĢ) Background fluorescence should be the same for the positive and negative controlģ) Compensation controls MUST match the exact experimental fluorochromeġ) Controls need to be at least as bright or brighter than any sample to which the compensation will be appliedĪn important consideration is to select the sample with the brightest fluorescence of the experiment. Three Rules for Compensation Controls (from TreeStar Daily Dongle )įirst and foremost, there must be a single stained control for every parameter in the experiment!
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