title: Identification of S-phase cells with PC10 antibody to proliferating cell nuclear antigen (PCNA) by flow cytometric analysis.
abstract: 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.
authors.affiliation: Department of Pathology, Iwate Medical University, Morioka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Beppu
authors.forename: Y
authors.initials: Y
authors.lastname: Ishida
authors.forename: H
authors.initials: H
authors.lastname: Arai
authors.forename: T
authors.initials: T
authors.lastname: Wada
authors.forename: N
authors.initials: N
authors.lastname: Uesugi
authors.forename: K
authors.initials: K
authors.lastname: Sasaki
mesh_terms: 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*
chemical_list: D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D001973:Bromodeoxyuridine