Measurement of Lymphoproliferation at the Single-Cell Level by Flow Cytometry

The recognition phase of the cell-mediated immune response is studied in vitro by monitoring responses of CD4⁺ T cells, cultured in the presence of specific antigen. Conventional cultures include mononuclear cells separated from peripheral blood by a complex and time-consuming procedure. Several investigators (1 –7 ) have suggested the use of whole-blood cultures which is easier to perform and involve less manipulations, thus reducing the risk of contamination, preactivation, or selective depletion or enrichment of cell subsets. The cell-mediated immune response involves a broad spectrum of measurable mechanisms. Multiparameter flow cytometry analysis permits the simultaneous detection of cell surface and intracellular molecules, as well as light-scatter characteristics of individual cells. In this chapter, a method is described for the measurement of proliferative responses in whole-blood cultures, using flow cytometric detection of lymphoblast development. The method is simple and reproducible, and can be combined with immunostaining for the detection of cytokine production, cell-surface expression of costimulatory molecules, and DNA synthesis.