This accuracy can be compromised when the signal in the experimental sample is brighter than the signal from the compensation control for a given channel.īy nature, dimly fluorescent populations tend to spread out (have higher coefficients of variation). The accuracy of the slope of the line described above is only as robust as the quality of the compensation controls used to generate it. The slope, or angle, of this line defines the amount of compensation required to correct the spillover between the two channels on the plots (see Figure 1 below). 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.
COMPENSATION FLOWJO SOFTWARE
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. The fluorophore used must be the exact fluorophore (i.e.Autofluorescence should be the same for the positive and negative populations used for the compensation calculation in each channel.Each compensation control must be as bright as, or brighter, than the experimental stain.The Daily Dongle, contains a much-cited and useful post, Three Rules For Compensation Controls and answers this question with three guidelines: What does it mean for a compensation control to be “proper” or “good”? What Is A High Quality Compensation Control? Given all of these points, the most important aspect of setting up a relevant, proper, and functional compensation matrix is to ensure that the spillover coefficient is accurate, is wholly reliant on good controls. This relationship between two detectors, the spillover coefficient, defines the spillover regardless of the amount of dye, so it can be used correctly for the spectral overlap in a typical experimental setting in which amounts of dye can vary widely between and within samples. fluorophore B) is defined by the ratio of A’s signal in B’s detector to A’s signal in its own detector. The compensation calculation relies on the fundamental concept that the amount of spillover of a fluorophore (e.g. This percentage corresponds to the amount of signal spillover signal that is contributed to this detector by all the other fluorophores being used in the experiment. The more colors measured within a single experiment, the more crowded the spectrum becomes and the more severe this kind of crosstalk is.Ĭompensation is a mathematical process that deals with this problem by removing a percentage of the total signal from each detector. F luorescence compensation is not possible without proper controls, so it is critical to spend the time and effort to generate high-quality controls in the preparation of an experiment.įirst, recall that compensation is a consequence of spectral overlap, which occurs because the fluorescence emission spectra of essentially all fluorophores are wider in wavelength range than the optical filters that we use to measure those fluorophores.īecause of this, fluorophore emission will often overlap with more than one filterset on the cytometer, leading to the detection of the signal from one fluorophore by multiple detectors (i.e.
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