Cell viability was assessed using the cell counting kit-8 (CCK-8). Briefly, the MSCs were cultured in 96-well plates at a density of 1,000 cells/well. Following an ~70% fusion of the cells, the indicated treatments were carried out. Subsequently, 10 μl CCK-8 solution were added to each well and the plates incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader (Tecan Infinite M200 microplate reader; LabX, Midland, ON, Canada). The mean optical density (OD) of 4 wells in each group was used to calculate the percentage of cell viability. The experiments were carried out in triplicate.
In order to determine the cell proliferative ability, the MSCs were cultured in 96-well plates for 24 h, followed by exposure to various concentrations of nicorandil for the indicated periods of time. An aliquot of 5-ethynyl-2′-deoxyuridine (EdU; 50 μM; Ribobio, Guangzhou, China) was added to the culture medium for 2 h. The cells were washed with PBS, fixed with 4% paraformaldehyde, and dyed with Apollo staining reaction liquid (Ribobio). Hoechst 33342 (Ribobio) was used for the labeling of the nuclei. Images were acquired using a fluorescence microscope. Five fields were randomly selected from each dish, and at least 3 dishes were counted per concentration.
In order to determine the cell proliferative ability, the MSCs were cultured in 96-well plates for 24 h, followed by exposure to various concentrations of nicorandil for the indicated periods of time. An aliquot of 5-ethynyl-2′-deoxyuridine (EdU; 50 μM; Ribobio, Guangzhou, China) was added to the culture medium for 2 h. The cells were washed with PBS, fixed with 4% paraformaldehyde, and dyed with Apollo staining reaction liquid (Ribobio). Hoechst 33342 (Ribobio) was used for the labeling of the nuclei. Images were acquired using a fluorescence microscope. Five fields were randomly selected from each dish, and at least 3 dishes were counted per concentration.