DoubletExclusion Gating
Measurement of particle or cell parameters by flow cytometry is predicated on the assumption that these measurements are made on single cells. Doublets and larger multiplets are most commonly detected by comparing fluorescence pulse peak height to pulse area. Using either a two-parameter histogram of pulse peak intensity versus pulse area and setting a bitmap around the singlet particles along the diagonal, or a single-parameter histogram of the ratio of pulse peak intensity to pulse area and setting a region around the singlet particle peak (Fig. 1.8.4), the user can restrict the analysis to the singlet particles by gating on the singlet particle gates (Snow and Bauer, 1994). This type of doublet-exclusion gating has become a routine part of DNA quantitation analysis. Any investigator designing an assay for which doublets or multiplets would significantly affect the results should consider this type of doublet-exclusion gating. The above-mentioned pulse
peak height versus pulse area may be applicable, or the alternative methods using pulse width (time-of-flight) or forward-scatter intensity may need to be considered.
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