Evaluating Intracellular Dehydrogenase Activity

The activity of the intracellular dehydrogenases was evaluated by using the Cell Counting Kit-8 (CCK- 8) (Sigma-Aldrich), a highly sensitive test that utilizes water-soluble tetrazolium salt [34 ,39 (link)]. WST-8 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt can be reduced by dehydrogenases in cells, producing an orange colored formazan dye soluble in the culture medium. The cells were counted by using Scepter™ 2.0 (Merck Millipore Corporation, New York, NY, USA) cell counter, seeded in 96-well plates at a density of 8 × 10³ cells/well, and pre-incubated for 24 h under standard conditions. Then, cells were treated for 72 h with different concentrations of the drug solution. Finally, the activity of the intracellular dehydrogenases was evaluated by adding CCK-8 solution (10 μL) to each well and measuring the absorbance at λ = 450 nm after 2 h of incubation with CCK-8 solution, using the microplate reader Victor^® 3V (PerkinElmer^®, Waltham, MA, USA). The changes of the cell viability were expressed as percentage (%) changes of intracellular dehydrogenase activity induced by the drug with respect to the control.