Evaluating AGE-Induced Cytotoxicity with LDH Assay

To determine the cytotoxicity of the AGEs, we performed lactate dehydrogenase (LDH) assay. LDH assay kit was obtained from Invitrogen. LDH production was determined as described previously [31 (link)]. Briefly, BV2 cells were treated with different concentrations of MGO-AGE and were incubated for 24 h. Conditioned medium (50 μL) from the treated cells were collected and mixed with equal amounts of LDH mixture (LDH substrate and buffer). This resulted in a color change into a brown-red color because of the conversion of lactate to pyruvate in the presence of LDH enzyme, which was evaluated my measuring the absorbance at 450 nm. This experiment was performed exactly as described by LDH assay kit protocol from Thermo Fisher Scientific/Invitrogen (catalogue no. C20300, Waltham, MA, USA).