Cell viability was measured 24 h after I/R using two different methods: The MTT assay, as a quantitative method, was performed as described previously [70 (link)]. Briefly, the cells were incubated with 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 10 min, which is metabolized by viable cells to a blue formazan derivative. Subsequently, the cells were lysed in 300 µL 0.04 M HCl in isopropanol and diluted with 300 µL destilled water. The absorbance was measured in a Tecan infinite M200 ELISA reader (Tecan, Männedorf, Switzerland) at 570 nm.
The Apoptotic/Necrotic/Healthy Cells Detection Kit (PromoKine, Heidelberg, Germany), a qualitative assay to analyze cell viability, was used to stain apoptotic and necrotic cells [43 (link)]: 200,000 to 300,000 cardiomyocytes were plated in 35 mm microdishes (Ibidi, Martinsried, Germany) for 10 to 14 days and the same number of fibroblasts were plated for three to four days. The cells were washed with PBS and incubated with hoechst33342, annexin V and ethidium homodimer III according to manufacturer’s instructions. Cells were visualized by laser scanning fluorescence microscopy using an LSM710 confocal microscope (Zeiss, Jena, Germany).
The Apoptotic/Necrotic/Healthy Cells Detection Kit (PromoKine, Heidelberg, Germany), a qualitative assay to analyze cell viability, was used to stain apoptotic and necrotic cells [43 (link)]: 200,000 to 300,000 cardiomyocytes were plated in 35 mm microdishes (Ibidi, Martinsried, Germany) for 10 to 14 days and the same number of fibroblasts were plated for three to four days. The cells were washed with PBS and incubated with hoechst33342, annexin V and ethidium homodimer III according to manufacturer’s instructions. Cells were visualized by laser scanning fluorescence microscopy using an LSM710 confocal microscope (Zeiss, Jena, Germany).