Ldh release detection kit

The cell viability was determined by MTT assays (Sigma-Aldrich Co.). Briefly, cells were seeded onto 96-well plates, and then 20 µL of MTT at a concentration of 5 mg/mL was added to the medium. The plates were placed for 4 hours in the dark at 37°C and 5% CO₂. After that, the medium was removed and 100 µL of dimethyl sulfoxide (DMSO) was added into the medium for 15 minutes in the dark at 37°C and 5% CO₂. Then, the samples were observed at a wavelength of 570 nm. The relative cell viability was recorded as a ratio to that of the control group. Apoptotic cells were quantified using a one-step TUNEL kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions.24 (link) Cells were seeded onto the 12-well plates and incubated with fluorescein–dUTP (Beyotime Institute of Biotechnology) for 30 minutes at 37°C in a 5% CO₂ atmosphere. After being labeled with DAPI, the cells were observed using a laser confocal microscope (TcS SP5; Leica Microsystems, Inc., Buffalo Grove, IL, USA). LDH was released into the medium when cellular membranes ruptured. To evaluate the levels of LDH in the medium, an LDH Release Detection kit (Beyotime Institute of Biotechnology) was used according to manufacturer’s protocol. Cells treated with PBS were used as the control group for MTT assay, LDH release assay, and TUNEL staining.