A flow cytometric assay for quantitation of rare antigen-specific T cells: using cell-tracking dyes to calculate precursor frequencies for proliferation.

Cell-tracking dyes stain cells with bright fluorescence which is partitioned between daughter cells after each cell division, so that the daughter cells have closely half the intensity of the parent cells from which they were derived. Therefore, the intensity of a cell, relative to its intensity at the time of staining, provides information about how many divisions have occurred. Knowing the number of division cycles that have occurred, one can calculate the number of cells in the original population (before culture) that were going to go on to proliferate. In this way, cell-tracking dyes provide a flow cytometric method for determining the proportion of cells in a population that will go on to proliferate in response to stimulation. This dye-dilution methodology can, therefore, detect proliferation of rare antigen-specific cells that increase in number during division and, importantly, can be used to back-calculate the precursor frequency of these rare cells. Simultaneously, the phenotype of these cells can be determined, as well as their ability to synthesize cytokines, and to express or not relevant antigen-binding receptors and activation markers.