Simultaneous analysis of surface marker expression and cell cycle progression in human peripheral bloods mononuclear cells

One method for examining cell cycle kinetics by flow cytometry uses continu ous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA flu orochrome Hoechst 33258. After counterstaining with a secondary DNA fluoroc hrome (e.g., ethidium bromide), the analyst can distinguish cells in differ ent phases of the cell cycle over a number of mitotic cycles with flow cyto metry. In this report, we describe a modification of the flow cytometric Br dU-Hoechst assay that allows combined analysis of cell proliferation and im munophenotyping at the single cell level. To demonstrate an application of this method, human peripheral blood mononuclear cells were stimulated with tetanus toxoid or interleukin-2 for up to 6 days in the presence of BrdU, h arvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD1 4, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker e xpression, for the purpose of immunophenotyping and identifying specific ce ll subsets responding to antigen stimulation. Our successful application of this method suggests that it may be used to study immune responses at the molecular and cellular level and to identify mechanisms of immune system mo dulation. (C) 2001 Elsevier Science B.V. All rights reserved.