Ldh cytotoxicity assay

The death of cells (2 × 10⁵ cells/well) with different treatments was evaluated with an LDH Cytotoxicity Assay (Beyotime, Beijing, China) following the instructions of manufacturer [1 (link)]. The results were calculated with the following formula: % cytotoxicity = (infected LDH − control LDH)/(max lysis LDH − control LDH) × 100%. Qualitative data were expressed as mean ± standard error of mean value (SEM; n = 3 or 6).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) cytotoxicity assay (Beyotime, Nanjing, China) were used to detect cardiomyocytes proliferation and lactate dehydrogenase release in the different groups according to the manufacturer's instructions. For MTT assay, cardiomyocytes were harvested at 48 h, after which 200 μl of MTT solution was added to each well. After 4 h of incubation at 37°C, the MTT solution was carefully aspirated and 200 μl of DMSO was added per well. The optical density of each well at 490 nm was read with a microplate reader (Molecular Devices, USA). The cell viability was calculated as follows: [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100. For LDH assay, the supernatants of cells were collected for the measurement of LDH release by a commercial LDH cytotoxicity assay kit (Beyotime, Nanjing, China). The absorbance at 490 nm was read using a microplate reader (BioTek Instruments, Inc.).

The cytotoxicity of NK cells was conducted by lactate dehydrogenase (LDH) cytotoxicity assay (Beyotime, China), according to the manufacturer’s instructions. Briefly, we collected cell supernatants which cocultured NK cells and A549 and H1299 cells for 4 h. Then, we performed LDH assay. The formula of cytotoxicity was calculated: Cytotoxicity (%) = [(OD_Experimental value–OD_Spontaneous value)/(OD_Maximum value–OD_Spontaneous value)] × 100 (Bian et al., 2020 (link)).