USE OF A MEMBRANE-LOCALIZED GREEN FLUORESCENT PROTEIN ALLOWS SIMULTANEOUS IDENTIFICATION OF TRANSFECTED CELLS AND CELL-CYCLE ANALYSIS BY FLOW-CYTOMETRY

The simultaneous detection of the green fluorescent protein (GFP) and DNA content using propidium iodide (PI) by flow cytometry is made diff icult because of the unique nature of these 2 fluorogenic reagents, Fo r PI to enter cells efficiently and to stain DNA quantitatively, the c ells must first be permeabilized; ethanol treatment is a routine metho d to achieve this, However, this permeabilization step causes GFP, whi ch is normally found in the cytoplasm, to leach out of the cells. Alth ough, the use of paraformaldehyde-based fixatives allows GFP to be mai ntained in cells acid retain its fluorescence even after ethanol perme abilization, the protocol we commonly employ results in inefficient PI staining and poor quality DNA histograms, To circumvent these difficu lties, we have employed a GFP-fusion protein which localizes to the ce llular membrane and as such is retained in cells upon ethanol permeabi lization without prior fixation. This allows the GFP signal to be dete cted in cells treated with ethanol in preparation for PI staining and cell cycle analysis, This property facilitates the use of GFP as a mar ker for transfected cells in experiments designed to characterize the effects of ectopic expression of cellular or viral genes on cell cycle progression. (C) 1997 Wiley-Liss, Inc.