Flow cytometric analysis of the cell cycle in transfected cells without cell fixation

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 U2OS and NIH 3T3 cells.