Cell survival assay using calcein acetoxymethyl (AM)

To assess cell viability, the calcein assay was performed with neonatal rat cardiomyocytes and adult mouse cardiomyocytes that were seeded in 96-well (1 × 8 Stripwell; 9102, Life Sciences) after H/R (24 h Hx and 24 h reoxygenation in neonatal rat cardiomyocytes and 12 h Hx and 6 h reoxygenation in adult mouse cardiomyocytes), with the use of Cell Counting Kit F (CK-06, Dojindo) as described previously.^18,23 Briefly, after being washed with PBS, cultured cells were incubated in calcein AM solution diluted in PBS (1:500 ratio of calcein AM to PBS) for 15 minutes at 37 °C. Thereafter, cell viability was fluorometrically measured (excitation wavelength of 490 nm and emission wavelength of 520 nm) with the use of a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific).