Fluorochrome compensation

Compensation Compensation is the way a flow cytometrist corrects for the overlap between the fluorescence spectra of different flu-orochromes. Without compensation, fluorescence from a given fluorochrome may be included to some extent in the intensity value coming from a photodetector assigned to the detection of a different fluorochrome. Compensation can be electronic or can be applied by software during analysis of stored data. 

When fluorochromes are combined in a multiparameter assay, it is almost always necessary to correct for signals from overlapping portions of emission spectra that have not been eliminated by optical filtration. This is accomplished through a spectral compensation procedure that is performed according to instrument-specific instructions, and involves the adjustment of detector voltages to electronically subtract extraneous fluorescence. 

A limitation of the flow cytometric binding assay has been the precise determination of the receptor affinity and calculation of the receptors per cell. This limitation appears to have been overcome by the development of fluorescein and phycoerythrin compensation-calibration standards (Flow Cytometry Standards Corp., Research Triangle Park, NC). These standards have made it possible to quantify the fluorescence intensity of samples labeled with fluorescein or phycoerythrin, and relate the intensity to molecules of equivalent soluble fluorochrome. These standards have been utilized in quantitative studies of neutrophil chemoattractant-ligand interaction (4). 

The analysis of a complex population of cells may call for the identification of the cells of interest with one MAb, and quantitation of antigen expression on these cells with a second antibody. The choice of fluorochrome for each purpose may be influenced by relative levels of expression of the markers involved, as well as the factors referred to above. There is a degree of spectral overlap between fluorochromes, and this may make sensitive quantitation of a low level antigen difficult in the presence of high level labeling of the first antibody used for cell identification. This can be mitigated to some extent by selection of the more sensitive fluorochrome (e.g., phyco-erythrin) for the low level quantitation. In any event, quantitative analysis requires careful adjustment of spectral overlap an adjustment procedure for multiple fluorochromes appears in Chapter 34, and quantitative adjustment of compensation is illustrated in Section 3.4. 

It remains to be said (unfortunately) that the compensation values are valid only for a particular pair of fluorochromes with a particular set of filters and mirrors and with particular voltages applied to a particular set of PMTs. If any one of those elements is altered, the required compensation values will alter as well. In general, once compensation has been set using single-stained particles with a given experimental protocol (PMT voltages, filters, and so forth), compensation values between a given pair of fluorochromes should remain... 

Cross-talk Cross-talk is the signal from the wrong photodetector that results because the fluorescent light emitted by one fluoro-chrome contains some light of a wavelength that gets through the filters on a photodetector that is nominally specific for the fluorescence from a different fluorochrome. See Compensation. 

Compensation between instrument channels is required where the emission spectra of the fluorochromes overlap. Compensation is necessary between channels 1 and 2 and between channels 2 and 3 but is not required between channels 1 and 3 because these spectra should not overlap. To set compensation use the compensation parameter menu and cell samples of known positivity for the fluorochromes that are being used, in this case known dead cells and known infected cells, and also negative controls. 

A number of methods for the study of apoptosis and necrosis by flow cytometry have been developed (see review by Steensma et al., 2003). Unfortunately, none of the methods are rigorously specific for apoptosis and many show poor overall specificity for cell death or cannot discriminate between the terminal stages of apoptosis and necrosis. Moreover, some of the fluorochromes used to detect apoptosis have emission spectra that fully overlap the spectra of those typically used for immunophenotyping or have a broad emission spectrum that makes compensation in multi- or polychromatic flow cytometry difficult, if not impossible. Thus, the preferred method would be one that measures two aspects of apoptosis, can discriminate between apoptotic and dead cells, and uses fluorochromes that allow simultaneous immunophenotyping. One such method is the annexin-V/7-amino-actinomycin-D assay described by Merchant et al. (2001) that is available in kit from BD Biosystems (San... 

As these two fluorochromes have nonoverlapping emission/excita-tion spectra, there is no need to have extra tubes with just one of the antibodies for compensation. 

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