The use of CFSE-like dyes for measuring lymphocyte proliferation: Experimental considerations and biological variables

The measurement of CFSE dilution by flow cytometry is a powerful experimental tool to measure lymphocyte proliferation. CFSE fluorescence precisely halves after each cell division in a highly predictable manner and is thus highly amenable to mathematical modelling. However, there are several biological and experimental conditions that can affect the quality of the proliferation data generated, which may be important to consider when modelling dye dilution data sets. Here we overview several of these variables including the type of fluorescent dye used to monitor cell division, dye labelling methodology, lymphocyte subset differences, in vitro versus in vivo experimental assays, cell autofluorescence, and dye transfer between cells.