title: Flow cytometric analysis of the cell cycle in transfected cells without cell fixation.
abstract: We describe a simple and rapid protocol for the flow cytometric analysis of the cell cycle in transfected cells using a green fluorescent protein anchored in the intracellular membranes of the endoplasmic reticulum (ER-GFP) as a transfection marker. The transfected cells are analyzed by dual-parameter flow cytometry after a brief incubation with digitonin, followed by staining with propidium iodide. Treatment of cells with digitonin efficiently preserves the ER-GFP fluorescence and allows reproducible and quantitative DNA staining, thus obviating the need for cell fixation before flow cytometry. The digitonin-based protocol is faster and easier to perform than conventional cell fixation and is illustrated herein by cell cycle analyses of U20S and NIH 3T3 cells.
authors.affiliation: Department of Molecular Genetics, University of Illinois College of Medicine, Chicago 60607-7170, USA.
authors.forename: D G
authors.initials: DG
authors.lastname: Pestov
authors.forename: M
authors.initials: M
authors.lastname: Polonskaia
authors.forename: L F
authors.initials: LF
authors.lastname: Lau
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002453:Cell Cycle / Q000235:genetics*; D004072:Digitonin; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D015415:Biomarkers; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004072:Digitonin