Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle.
Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.
Landberg
G
G
W.M. Keck Autoimmune Disease Center, Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, California.
Tan
E M
EM
Roos
G
G
D000951:Antigens, Neoplasm / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis*; D002453:Cell Cycle; D002460:Cell Line; D004273:DNA, Neoplasm / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D019394:Ki-67 Antigen; D007700:Kinetics; D009687:Nuclear Proteins / Q000032:analysis*; D018809:Proliferating Cell Nuclear Antigen; D014407:Tumor Cells, Cultured / Q000166:cytology
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D004273:DNA, Neoplasm; D019394:Ki-67 Antigen; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen