Cell Viability Evaluation Using Fluorescent Stains

Cellstain Double Staining Kit (Dojindo, Tokyo, Japan) was used to test cell viability as described previously [9 (link)]. Briefly, seeded cells were stimulated according to specific conditions. Afterward, propidium (4 μM) [12 (link)] and calcein-AM (2 μM) were used to stain cells. Ten minutes later, the live and dead cells were discriminated. The living cells hydrolyze calcein-AM by intracellular esterase, generating green fluorescence. In contrast, the dead cells showing red fluorescence. The cell images were recorded with an Olympus CCD camera attached to the immunofluorescent microscope (IX71S1 F-2; Olympus, Tokyo) under the identical settings.