Lactate Dehydrogenase (LDH) Leakage Assay and Determination of ATP Content

Lactate dehydrogenase (LDH)-release assay was used to detect the cytotoxicity of psoralen. Cells were seeded into 24-well plates, and then treated with DMSO or psoralen. 10X lysis solution was used to generate a maximum LDH release as the positive control wells. LDH-leakage level was measured via transferring 50 μL culture supernatants from all of the wells to a fresh 96-well plate and the subsequent processes were performed according to the instructions of CytoTox 96^® Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI, United States). The percent cytotoxicity = 100 × Experimental LDH Release (OD490)/Maximum LDH Release (OD490). In addition, the cells at the bottom of 24-well plate were used to detect the intracellular ATP content according to the manufacturer’s protocol of CellTiter-Glo^® 2.0 Assay Kit (Promega). The ATP content was normalised against the cellular TP content, which was quantified using the BCA Protein Assay Kit (Beyotime, Nanjing, China). The mitochondria of liver tissues were isolated using the Tissue Mitochondria Isolation Kit (Beyotime), and the ATP and protein content were measured using the same method as above.