2.6. MTS Assay

The MTS (CellTiter 96^® AQueous One, Promega, Fitchburg, WI, USA) assay was performed following the manufacturer’s recommendations. The tetrazolium compound MTS was bio-reduced by cells into a colored formazan product that is soluble in tissue culture medium [20]. This conversion was presumably accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active (viable) cells.

After the incubation period, media containing the chemicals were removed from all of the wells, and cells were washed with 150 μL/well of prewarmed PBS. In turn, 100 μL of DMEM/F-12 (without phenol red, L-glutamine, or HEPES) was added to each well, in order to eliminate the absorbance signal given by the pH indicator; 20 μL of CellTiter 96 ^® AQueous One Solution Reagent (Promega, Fitchburg, WI, USA) per well was also added. The CellTiter 96 ^® AQueous One Solution Cell Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation assays or cytotoxicity. To blank wells, PBS was added. The cells were incubated for another 4 h. Finally, absorbance was detected at 490 nm (as well as at 650 for noise elimination) using a monochromator microplate reader safire2 (Tecan Austria GmbH, Salzburg, Austria)/ measurement parameter editor Magellan (version 6).