![]() The spillover coefficient defines a ratio of interaction between two channels that is independent of the amount of fluorophore.Īnother way of thinking about the spillover coefficient is that it defines the slope of a line that can be drawn between the nonfluorescent and fluorescent populations, connecting their medians, in an uncompensated plot of data from a single-color control. The answer is a firm no: they don’t need to be matched. You may be wondering whether the compensation matrix may be irrelevant and inaccurate if compensation controls signal intensities are not exactly matched to the experiment’s samples. 3 Keys To Creating High Quality Compensation Controls 1. Account for brightness. Regardless, when using a universal negative, the operator instructs the cytometry software to utilize the nonfluorescent population in the universal negative control and to ignore any nonfluorescent cells or beads in the single-stain controls. Note that the single-stained controls may or may not contain negative cells. In the latter case, the controls would consist of (1) a single universal negative tube, used to designate the negative population in all channels and (2) a tube containing stained cells or beads for each channel. a lysed whole blood stained with a marker which will be present on only a portion of the total number of cells) Keep in mind that properly compensating a channel requires both a fluorescent, or stained population (for every fluorescent channel), and a nonfluorescent,or very dim, population. same molecular structure) that is used in the experimental sample.
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