A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using perip heral blood mononuclear cells responding to tetanus toroid, we describe an extension of this dye methodology to calculate the precursor frequency of a ntigen-specific T-cells. With mathematical deconvolution of the fluorescenc e histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor f requency of cells in the original population that responded to the specific stimulus, Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for pre cursor frequency. Based on the characteristics of flow cytometric data acqu isition, it is estimated that the now method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10 (5) of the population. When comparing results to those from the limiting di lution technique, the flow cytometric method returns values that indicate h igher precursor frequencies. Possible reasons for this discrepancy are disc ussed. The flow cytometric method offers the advantage of simplicity as wel l as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, rou tine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy. (C ) 1999 Elsevier Science B.V. All rights reserved.