Cell viability measurement

IEC-6 cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Sigma-Aldrich). After either a 4- or 24-h exposure to the nanoparticles, CuCl or CuSO₄, the media was removed and the cells were washed 3 times with phosphate buffered saline (PBS) (37 °C) and replaced with 100 μL media and 20 μL MTS. The plates were returned to the incubator for 1–4 h, and then absorbance was measured at 490 nm with a SpectraMax i3 (Molecular Devices, Sunnyvale, CA) plate reader.

Viability of the human intestinal model was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT is the recommended viability assay by the vendor for this model. After either a 4- or 24-h exposure to the NPs or CuSO₄, media was aspirated, and the cells were washed 3 times with PBS (37 °C). After the addition of 300 μL of MTT solution to each well, the plates were returned to the incubator for 3 h. Excess MTT solution was removed and 1 mL per well of isopropanol was added to extract the formed formazan (reduced product of MTT) from the cells. After a 1 h extraction, an additional 1 mL of isopropanol was added and each well was mixed thoroughly before absorbance was measured at 570 nm with the plate reader. (Note: we began our studies with the rat IEC-6 cells and chose to use the MTS assay because it does not require an extraction step of the reduced formazan. We moved onto the human EpiIntestinal model and the manufacturer recommended that the MTT assay be used to measure viability.)

Nanoparticles have been noted to interfere with some cell viability assays (Holder et al., 2012; Petersen et al., 2014). The cells were washed with PBS three times before addition of MTS or MTT, which should remove non-absorbed NPs. Incubating CuO NPs (80 μg Cu/mL) with MTS or MTT resulted in no color change of the tetrazolium compounds (i.e., reduction). CuO NPs did not inhibit the direct reduction of MTS or MTT by ascorbic acid (1 mM).