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 anch ored in the intracellular membranes of the endoplasmic reticulum (ER-GFP) a s a transfection marker: The transfected cells are analyzed by dual-paramet er flow cytometry after a brief incubation with digitonin, followed by stai ning with propidium iodide. Treatment of cells with digitonin efficiently p reserves the ER-GFP fluorescence and allows reproducible and quantitative D NA staining, thus obviating the need for cell fixation before flow cytometr y. The digitonin-based protocol is faster and easier to perform than conven tional cell fixation and is illustrated herein by cell cycle analyses of U2 OS and NIH 3T3 cells.