Simultaneous analysis of surface marker expression and cell cycle progression in human peripheral blood mononuclear cells.
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.
Rosato
M T
MT
Consortium for Risk Evaluation with Stakeholder Participation, University of Washington, Seattle, USA.
Jabbour
A J
AJ
Ponce
R A
RA
Kavanagh
T J
TJ
Takaro
T K
TK
Hill
J P
JP
Poot
M
M
Rabinovitch
P S
PS
Faustman
E M
EM
D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D006690:Bisbenzimidazole / Q000737:chemistry; D001973:Bromodeoxyuridine / Q000737:chemistry; D002453:Cell Cycle; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000737:chemistry; D004996:Ethidium / Q000737:chemistry; D005404:Fixatives / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008213:Lymphocyte Activation; D016131:Lymphocyte Subsets / Q000737:chemistry / Q000145:classification* / Q000166:cytology; D011108:Polymers / Q000737:chemistry
D000944:Antigens, Differentiation, B-Lymphocyte; D000945:Antigens, Differentiation, T-Lymphocyte; D005404:Fixatives; D005456:Fluorescent Dyes; D011108:Polymers; D003609:Dactinomycin; D005557:Formaldehyde; C025942:7-aminoactinomycin D; D004247:DNA; D004996:Ethidium; D001973:Bromodeoxyuridine; D006690:Bisbenzimidazole; C003043:paraform