MTT viability assay

An MTT assay was performed using the MTT Cell Count Kit (Nacalai Tesque, catalog no. 23506-80) following the manufacturer's specifications. Briefly, 10000 cells were seeded in a 96 well dish and left to incubate for 48 h. The medium was replaced to phenol free D-MEM containing the correct concentration of glucose, and cells were again left to incubate for 48 h. Following this, 10 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was pipetted into each well before incubating the plate for 3 hours at 37°;C and at controlled CO₂ concentrations_. The reaction was stopped with 100 μL of acidified isopropanol (0.04 M HCl in absolute isopropanol), and a colorimetric reading was taken at 595 nm.