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. Compensation controls MUST match the exact experimental fluorochromeĬontrols need to be at least as bright or brighter than any sample the compensation will be applied toĪn important consideration is to select the sample with the brightest fluorescence of the experiment.Background fluorescence should be the same for the positive and negative control.Controls need to be at least as bright or brighter than any sample the compensation will be applied to.In addition, there are three rules for “good” compensation controls: It automates the manual process to increase efficiency and reduce user error.įirst and foremost, there must be a single stained control for every parameter in the experiment! This is done with a sophisticated algorithm that calculates the ratio of the difference in spillover fluorescence divided by the difference in the primary fluorescence for every combination of single stain control. Software compensation is much easier because all that is required is to identify the positive and negative populations in each single stain control and the software can calculate the MFI values and amount of spillover automatically. Imagine adding a third or even fourth parameter.
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