2.9. Cell Viability as Measured by the MTT Assay

MTT is an assay developed to determine viability based on cellular metabolic activity. Cells were treated on day 5 post-seeding with the various concentrations of Folic Acid equivalent to Selenofolate for 6 days. Control treatments included 20 µM selenite/well and PBS alone. Phenol red-free DMEM—high glucose, + 10% FBS and MTT at 5 mg/mL was sterilized through a 0.22-micron filter, protected from light exposure, and used immediately. A 10% (v/v) solution of 50 µL of MTT (5 mg/mL) was added to each well and samples were incubated at 37 °C for 3 h. Following incubation with the MTT, 500 µL formazan solubilization solution and acidified isopropanol (0.1N HCl) with 10% Triton X-100, was added in each well. To assess metabolic activity, a Cytation 3 plate reader (BioTek, Winooski, VT, USA), at 570 nm absorption with subtraction of the 690 nm absorption, was used to quantify dissolved Formazan. The control cells were considered to be 100% viable and cell viability of the treated cells were calculated as a percentage of control cell absorbance as shown in Equation (1).

Equation (1). Determination of Cell Viability Using MTT.