(Authors):Cytomation,Inc. Address
A simple, rapid method of determining the percentage of viable cells within a test population is described using Hoechst 33342 (HO) dye. This technique can be performed using economical arc-lamp-based flow cytometers and requires the collection of only a single fluorescence parameter. In the current procedure, cell suspensions are maintained at 4oC and are analyzed within 5 minutes of the addition of HO (1ug /ml final). Non-viable cells show an immediate uptake of the dye and appear as a relatively bright population upon analysis. Viable cells, which incorporate the dye at significantly reduced rates appear relatively dim upon analysis. The separation of these populations is greater than one full decade on a single parameter, logarithmic histogram. It should be noted that dim, viable cells are still sufficiently bright to trigger single parameter cytometers which do not collect light scatter characteristics. HO-derived viability data from samples with varying proportions of viable and non-viable cells correlated well with data obtained by propidium iodide staining. This technique may therefore be used in multi-laser flow cytometers as a substitute for propidium iodide and thus frees long wavelength detectors for additional analyses in multicolor experiments.