Analysis with flow cytometry of green fluorescent protein expression in leukemic cells

BACKGROUND: The measurement of DNA content with propidium iodide (PI) in cells transfected with expression vectors encoding the green fluorescent protein (GFP) is a useful tool in studying a variety of biological functions of proteins within cells. The purpose of this study was to determine conditions of formaldehyde fixation that permit intracellular GFP fluorescence and adequate DNA histograms to be generated following transient transfection of cells with a GFP-encoding plasmid. Cell cycle analysis was also performed in GFP-positive cells. METHODS: The murine myeloid leukemic cell line, 32Dcl3, was used as the model system. Cells were transfected with a GFP-encoding plasmid (pEGFPC1). Following fixation in different formaldehyde concentrations and permeabilization with 70% ethanol, cells were stained with PI and analyzed by flow cytometry for GFP fluorescence and DNA content. Transfected cells were also analyzed for GFP fluorescence and DNA content following release from nocodazole block. RESULTS: Fixing cells in 0.51-1.75% formaldehyde concentrations prior to ethanol permeabilization resulted in 14-19% of transfected cells being GFP-positive, with acceptable coefficients of variation on the G(1) peak of DNA histograms. Analysis of cells synchronized to and released from the G(2)-M phase by nocodazole suggested that GFP-positive cells, when compared to GFP-negative cells, did not appear to progress out of G(2)-M following release from nocodazole block. Simultaneous detection of GFP fluorescence and DNA content by PI staining is possible following transient transfection of cells with a single expression vector encoding GFP. Our results demonstrate that GFP expression can be detected, using flow cytometry to perform cell cycle analysis in murine leukemic cells.