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

• Published in: Journal of immunological methods Vol. 256; no. 1; pp. 35 - 46
• Main Authors: Rosato, Michael T, Jabbour, Abdallah J, Ponce, Rafael A, Kavanagh, Terrance J, Takaro, Timothy K, Hill, Juliane P, Poot, Martin, Rabinovitch, Peter S, Faustman, Elaine M
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.10.2001; Elsevier
• Subjects: Antigen stimulation; Antigens, Differentiation, B-Lymphocyte - analysis; Antigens, Differentiation, T-Lymphocyte - analysis; Biological and medical sciences; Bisbenzimidazole - chemistry; bromodeoxyuridine; Bromodeoxyuridine - chemistry; Bromodeoxyuridine incorporation; Cell Cycle; Cell Membrane Permeability; Cell proliferation; Cells, Cultured; Dactinomycin - analogs & derivatives; Dactinomycin - chemistry; Diverse techniques; DNA - analysis; Ethidium - chemistry; Fixatives - chemistry; Flow Cytometry; Fluorescent Dyes - chemistry; fluorochromes; Formaldehyde - chemistry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Hoechst quenching; Humans; Immunobiology; Immunophenotype; Immunophenotyping - methods; Lymphocyte Activation; Lymphocyte Subsets - chemistry; Lymphocyte Subsets - classification; Lymphocyte Subsets - cytology; Lymphoid cells: ontogeny, maturation, markers, receptors, circulation and recirculation; Microbiology; Molecular and cellular biology; Polymers - chemistry; Cell proliferation; PBS; Flow cytometry; Immunophenotype; Antigen stimulation; PHA; IL-2; PFA; Hoechst quenching; PBMC; 7-AAD; FITC; HO-58; PE; FBS; Bromodeoxyuridine incorporation; PI; Th1; EB; BrdU; Human; Incorporation; Extinction; Use; Thymidine; Method; Cell surface; Blood; Infection; Continuous process; Blood cell; DNA; Cell cycle; Bacteriosis; Kinetics; Tetanus; Pyrimidine nucleoside
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00428-8

One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in different phases of the cell cycle over a number of mitotic cycles with flow cytometry. In this report, we describe a modification of the flow cytometric BrdU–Hoechst assay that allows combined analysis of cell proliferation and immunophenotyping 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, harvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD14, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker expression, for the purpose of immunophenotyping and identifying specific cell 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 modulation.