Estimation of Cell Viability by Flow Cytometry

The estimation of the viability of a cell population by flow cytometry is based on a simple yet powerful principle: Dead cells leak. Cells that die via the necrosis pathway, in contrast with apoptosis, rapidly lose membrane integrity (1 ). All of the different methods for evaluating viability are based on either direct leak detection or measurement of a direct consequence of this leakage. Dyes are used that either do not leak into live cells or do not leak out of live cells. Some combination methods use two dyes of different colors and direction of leak detection. The advantage to this is that there are two independent measures of the same phenomenon. This reduces the probability of deceptive results. In addition, these methods more clearly indicate cells that are damaged or dying but are not yet completely dead. The disadvantage is the expenditure of two colors for viability reduces the availability of colors for simultaneous measurement of other cellular parameters. Some methods look at a consequence of cell leakage. For example, cells that leak are not capable of maintaining an electrical potential across the cell membrane. Therefore, potential-sensing dyes can be used to estimate viability, although this is primarily used with bacterial studies (2 ,3 ). In addition, cell-associated enzyme activity stops either because of loss of energy or direct leakage of the enzyme; therefore, loss of enzyme activity can be used as a different estimate of cell viability. For example, many dyes used to evaluate cellular ions are nonfluorescent acetomethoxyesters (AM) that are cleaved by nonspecific esterases into the fluorescent ion-sensitive compound.