Cell viability was evaluated by the MTT colorimetric technique [37]. Briefly, 20 μL of the yellow tetrazolium (MTT (3-(4,5-dimethylthiazol-2)-2,5 diphenyl tetrazolium bromide) (Sigma-Aldrich, Milano, Italy, 5 mg/mL in phosphate-buffered saline (PBS)), was added to each well. The plates were incubated for 3 h at 37 °C 5% CO2, for reduction of MTT by metabolically active cells. The medium was then carefully removed and solubilization of formazan crystals (insoluble purple product of MTT reduction) 200 μL DMSO (Dimethyl Sulfoxide) was added to each well. The plates were placed on a shaker for 15 min in order to achieve the complete solubilization of the crystals and then the optical density of each culture supernatant was determined. The quantity of formazan product was measured by the amount of 540 nm absorbance, which is directly proportional to the number of living cells in culture. Changes in cell viability measured by MTT were compared to that of PBMCs in complete RPMI culture medium used for preparing NPs suspensions and cells exposure.

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