Measuring cell viability with membrane impermeable zinc fluorescent indicator

Abstract

Recent findings suggest that the accumulation of cytoplasmic zinc [Zn2+]i is a ubiquitous component in the cell death cascade. Zn2+ can be liberated from intracellular stores following oxidative stress and contribute to cell death processes. Here we show that the membrane/cell impermeable Zn2+ fluorescent indicator Newport Green (NG), which is non-toxic and impermeable to the membranes of healthy cells, can label unhealthy cells in tissue slices in a manner comparable to the traditional viability indicator propidium iodide (PI). Using confocal microscopy, we detected PI labeled nuclei colocalized with NG fluorescence. Our results indicate that cells which absorbed PI into their nuclei also allowed cell-impermeable Zn2+ dye to penetrate their plasma membranes, subsequently exhibiting cytosolic and nuclear fluorescence. As in PI staining, we observed marked increases in NG fluorescence in damaged/dead cells of tissue slices. Two other cell impermeable fluorescent Zn2+ dyes, Fluozin-3 and Zinpyr-4, also stained cytosolic Zn2+ in PI labeled cells. Our data indicates that the application of a Zn2+ fluorescent indicator is a fast, simple, non-toxic and reliable method for visualizing cell viability within in vitro tissue preparations. Accordingly, we demonstrate that intracellular accumulation of Zn2+ correlates with neuronal death.