Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes

We describe an improved twin-probe multiparameter flow cytometric technique to examine cell membrane permeability. Ability to retain preloaded intracellular bis-carboxyethyl carboxy fluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) is measured, simultaneously with forward and right-angle scatter. This has significant advantages over an earlier method using fluorescein together with ethidium. In addition to the two expected cell populations which were stained green positive, red negative (by convention membrane “intact” and “viable,” Region 1) and green negative, red positive (“membrane-damaged” and “non-viable,” Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 2). This was true for each of 9 cell types in vitro. For EMT6 mouse mammary tumour cells held under sub-optimal conditions or treated with membrane-active drugs, progression from Region 1 to Region 2 was observed, followed by further progression from Region 2 to Region 3. Cells eventually accumulated in Region 3. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and in turn to extracellular propidium.