Evaluating Cell Viability in 3D HaCaT Spheroids

The cell viability was assessed by WST8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo Molecular Technologies, Japan), which, in the presence of an electron mediator, is reduced by dehydrogenases in cells (as a vitality biomarker) to formazan dye, which is soluble in the tissue culture medium. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. The decrease in absorbance between the treatment after 24 h and the control was monitored at 37 °C at 450 nm using an Allsheng FlexA-200 Microplate Reader [15 (link)]. For this set of experiments, BET-CA formulation was added to the medium of human 3D HaCaT spheroids for 24 h (range in content of malvidin: 0.05–1.1 μg mL⁻¹). The free radical-generating chemical menadione (50 μM for 16 h of treatment) was used as cytotoxic agent [14 (link)]. 24 h treatment with malvidin in BET-CA (range of 0.07–0.6 μg mL⁻¹) was used as a reference.