Cytotoxicity/necrosis (LDH) assessment

Following exposure, 50 µL of conditioned cell culture medium were removed and added to a blank 96-well plate (triplicate measurement per sample). Control cell lysate was utilized as a positive control for LDH leakage. Ten microliters of cell lysis buffer (Promega Corp., USA) were added to each of the lysis control wells and incubated for 30 min. Fifty microliters of cell lysis/conditioned media was added to the 96-well plate. Equal volume of lactate dehydrogenase (LDH) assay substrate (CytoTox-ONE Homogenous Membrane Integrity Assay G7891, Promega Corp., Madison, WI, USA) was added to each of the wells and incubated at 37 °C for 30 min. The fluorescence intensity was then measured with a SpectraMax GeminiXS micro-plate reader at 560 nm excitation and a 590 nm emission wavelengths. Results were plotted as % increase in LDH with respect to the control condition (0 µg/mL treatment concentration).