Identification of S-phase cells with PC10 antibody to proliferating cell nuclear antigen (PCNA) by flow cytometric analysis.
We estimated the expression of proliferating cell nuclear antigen (PCNA) in HeLa S3 cells by flow cytometry with monoclonal antibody (MAb) PC10. HeLa cells were fixed with six different fixation procedures: 15-min and 30-min acetone, 15-min acetone followed by 15-min methanol (acetone/methanol), 30-min methanol, 15-min methanol followed by 15-min acetone (methanol/acetone), and a mixture of acetone and methanol. The fixed cells were applied to MAb PC10 against PCNA and then treated with FITC. With five fixation procedures except for acetone/methanol, PCNA was expressed in almost all cells with similar shapes and different FITC intensity levels on PCNA/DNA bivariate cytograms, whereas acetone/methanol fixation allowed PCNA detection in S-phase cells with a cytogram that showed a horseshoe-like pattern with a peak level at mid-S-phase. Flow cytometric dual parameter analysis of PCNA/BrdU was carried out in HeLa cells to confirm detection of PCNA in S-phase cells with acetone/methanol fixation. The population of cells stained for both parameters, i.e., S-phase cells, was obviously discriminated from that of the non-S-phase cell in PCNA/BrdU bivariate cytograms. These results strongly suggest that PCNA used with acetone/methanol fixation would be equal to BrdU as an S-phase marker.
Beppu
T
T
Department of Pathology, Iwate Medical University, Morioka, Japan.
Ishida
Y
Y
Arai
H
H
Wada
T
T
Uesugi
N
N
Sasaki
K
K
D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine / Q000032:analysis; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis*; D018809:Proliferating Cell Nuclear Antigen; D016196:S Phase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D001973:Bromodeoxyuridine