Multiskan mcc plate reader

The viability of cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method with an in vitro toxicology assay kit from Sigma as described earlier [16 (link),17 (link)]. It was used to measure mitochondrial activity. Cells were seeded in 24-well plates with 500 µL of F12K medium for 24 h followed by switching to serum-free medium. After 24 h of treatment just before adding MTT, 100 µL supernant was removed to be used for LDH assay (Figure 1). MTT was added to each well for 2 h according to the protocol outlined by the manufacturer. After removing the supernatant, formazan crystals were dissolved by adding equal volume of solution. At the end of the treatment period, 300 μL of culture medium was removed from each well and 20 μL of MTT solution (5 mg/mL) was added and incubated for 30 min. After distribution to a 96-well plate, absorbance was measured at 595 nm with the Thermo-Fisher Multiskan MCC plate reader (Fisher).

The activity of lactate dehydrogenase (LDH) was measured using a lactate dehydrogenase activity assay kit (Sigma) as described earlier [16 (link),17 (link),18 (link)]. A volume from the MTT assay was used and plated in a 96-well plate. An LDH master mix was prepared and added to each well. The reaction was carried out at room temperature in the dark. The resultant absorbance was measured at 450 nm with the Thermo-Fisher Multiskan MCC plate reader (Fisher).