Assessment of cell viability using high molecular weight dyes such as trypan blue has become a widely used method to differentiate viable from damaged cells. 8,17
Normal cells or those that are in the early phase of apoptosis, have intact plasma membrane, and thus exclude trypan blue; whereas, dead cells or those that are in late apoptotic stages with lysed membranes are permeable to the dye. The trypan blue assay for cell viability is inexpensive and reliable and is thus one of the most commonly used viability assays. Both adherent as well as suspension cells can be easily stained with trypan blue and counted using a hemocytometer under a light microscope. Viable cells exclude the dye and appear unstained or white, whereas dead cells take up the dye and are stained blue.
The usefulness of viability assays using trypan blue, particularly for some cell types, is somewhat limited due to the fact that once cells are exposed to the dye, the number of dead cells tends to increase with incubation time. 18,19
As time goes on, the percentage of dead cells artificially increases during this assay. One can overcome this problem by incubating cell samples for different experimental time points with trypan blue immediately prior to counting it, thus ensuring reliable results.