2.13. Lactate Dehydrogenase (LDH) Release and ATP Assay

LDH release was quantified in the cell culture supernatant using the LDH Cytotoxicity Assay Kit (Beyotime Biotech), according to the manufacturer's instructions. Briefly, appropriate treatments were added according to the experimental needs when the cell density reached 80–90%. One hour before the scheduled detection time point, LDH release reagent was added to the sample, mixed by pipetting several times, and incubated. After reaching the predetermined time, the cell culture plate was centrifuged at 400 g for 5 min. An aliquot of supernatant (120 μL) was used to measure absorbance at 590 nm and 680 nm using a Synergy H1 microplate reader (BioTek Instruments). The fold increase in LDH concentration was normalized to the control.

ATP concentrations in the cell culture supernatant were determined using the Luminescent ATP Detection Assay Kit (Abcam) and Colorimetric/Fluorometric ATP Assay Kit (Abcam) following manufacturer protocols. Luminescence and Colorimetric/fluorometric absorbance were measured using a Synergy H1 microplate reader (BioTek Instruments Inc., Winooski, VT, USA).