4.3. Cell Viability Assay (MTS)

Np dilutions were prepared in 96 well U plates (Tissue Culture Plate, 96 well, U-Bottom, Falcon, Corning, NY, USA) in order to obtain concentrations of 25, 50, 100, 200, 400 and 800 µg/mL of Al₂O₃, CeO₂, TiO₂ and Y₂O₃ in the combination. These Nps were combined with ZnO Nps at the IC50 concentration, 60 µg/mL, and incubated with Jurkat cells. For each Np, three independent experiments were performed.

The cell viability assays were carried out at a density of 6 × 10⁴ Jurkat cells per well in a 96-well plate (Tissue Culture Plate, 96-well, Flat-Bottom, Falcon) and the cells were left in the incubator for 24 h prior to the addition of the Nps. The cells were subsequently incubated with the combination of Nps for a further 24 h.

Once the incubation had finished, a colorimetric cell viability assay was performed using the Cell Titer 96^® AQueous One Solution Cell Proliferation Assay kit (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions.

The supernatant was removed from the plates by centrifugation at 600× g for 1 min at 4 °C (Sigma 2-16 KL Sartorius, Göttingen, Germany). The MTS stock solution was then diluted 1:6 (v/v) in cell culture medium and 120 µL of the diluted solution per well were added. The plates were incubated for 1 h and centrifuged again. Afterwards, 100 µL of the supernatant per well were placed in a clean plate in order to remove possible interferences caused by Nps.

In order to obtain a positive control for cell death, 100 µL of 5% Triton X-100 (Sigma-Aldrich, Steinheim, Germany) were added to some wells 1 h before the addition of the diluted MTS solution. RPMI culture medium and Nps alone were used as negative controls for cells alone and cells with NPs, respectively. Finally, absorbance was measured at 490 nm on an Envision multidetector (Perkin-Elmer Inc., Norwalk, CT, USA). The MTS assay is based on the conversion of a tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] into a coloured and soluble product in the culture medium, namely formazan. This coloured product results from the mitochondrial activity of living cells. The quantity of formazan produced by dehydrogenase enzymes, measured at 490 nm, is directly proportional to the number of living cells. Cell viability, expressed as a percentage, was calculated as follows:

[A]_treatment is the absorbance of the cells incubated with the Nps minus the absorbance of the Nps, and [A]_control is the absorbance of the untreated cells minus the absorbance of the culture medium.