2.7. MTT cell viability and LDH cytotoxicity assays

ARPE-19 cells were transfected with scramble non-targeted control, miR-144-5p or miR-144-3p mimic or inhibitor (antagomiR) and treated with 200 μM t-BHP. After different time intervals (0–4 h), culture media was replaced with 100 μl MTT (Sigma-Aldrich, St. Louis, MO) solution (0.5 mg/ml in culture media) and incubated at 37 °C. After 3 h, MTT solution was discarded; all wells were washed with PBS and 100 μl DMSO was added to each well. The absorbance was read at 540 nm (VersaMax Microplate Reader, Molecular Device, Sunnyvale, CA) and data was expressed as percent cell viability.

The amount of lactate dehydrogenase (LDH) released in the cell culture media was assessed using Pierce LDH Cytotoxicity assay kit as per manufacturer’s instruction (Thermo Scientific, Wilmington, DE). In 96-well plates, ARPE19 cells were treated as mentioned above. At the end of incubation, 50 μl cell culture media from the different wells was mixed with 50 μl reaction mixture and incubated in dark at room temperature. After 30 min, 50 μl stop solution was added and absorbance was read at 490 and 680 nm on a VersaMax Microplate Reader (Molecular Device, Sunnyvale, CA). LDH release is presented as percent cytotoxicity, which was calculated per the manufacturer’s instruction.