Lactate Dehydrogenase Assay

Cell death after MPA treatment was assessed using the Pierce^™ lactate dehydrogenase (LDH) Cytotoxicity Assay (Thermo Fisher Scientific, Waltham, MA, USA) kit. Reaction substrates were prepared as per the manufacturer’s instructions. LDH assay was performed with the media from the black-walled clear bottom 96 well plate. Forty-five minutes before the end of the 72-h exposure period, 10 μl 10× lysis buffer was added to one control well, and the plate was placed back in the incubator. After the exposure period, 50 μl of media was carefully removed from each well and transferred to a new 96 well clear-bottom assay plate. Next, 50 μl of the LDH reaction mixture was added to each well, and the plate was incubated for 30 min at room temperature, protected from light. The reaction was stopped by adding 50 μl of LDH stop solution to each sample. The plate was read using the BioTek Synergy H1 Hybrid Reader (BioTek) at an absorbance of 490 nm and 680 nm.