2.4.3. Cell Viability Assays

After 24 h of treatment, the medium from J774A.1 and BV2 treated and non-treated (control) cells was removed, and 100 μL of fresh culture medium containing 500 μg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or 50 μg/mL of Neutral Red (NR) was added to each well to access cell viability. The MTT assay involves the conversion of the water soluble MTT (yellow) by mitochondrial dehydrogenases in viable cells to a water-insoluble formazan (purple/blue) [24] while the NR assay is based on the ability of viable cells to incorporate and bind the dye neutral red in the lysosomes [25]. In both assays, the intensity of the color is directly proportional to cell viability. The cells with MTT or NR loading medium were incubated for at 37 ◦C in a 5% CO2-supplemented atmosphere. After 30 min, the respective loading medium was removed, and the attached cells were gently washed once with PBS. To solubilize the formazan crystals, 100 μL/well of dimethyl sulfoxide (DMSO) was added and the absorbance was measured at 570 nm using a microplate reader (Synergy H1 Hybrid Multi-Mode, BioTek/Agilent, Winoosky, VT, USA). To extract NR dye from lysosomes, 100 μL of acidified ethanol (1% glacial acetic acid, 50% aqueous ethanol) was added and the plate was placed on the plate shaker for ~10–15 min with protection from light. The absorbance at 540 nm and at 690 nm was measured in the microplate reader within 60 min of adding NR Desorb solution. For calculations, 690 nm was subtracted from 540 nm. The non-treated cells (control) were considered 100% of viable cells in both MTT and NR assays. For statistical significance, both cytotoxicity assays were repeated at least 3 times in triplicates.

The LIVE/DEAD® viability assay is a quick and easy two-color assay to determine the viability of cells in a population. Typical results will distinguish live cells which are stained with green-fluorescent calcein-AM to indicate intracellular esterase activity, from dead cells that are stained with red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. The assay was performed according to the manufacturer's instructions. Briefly, after 24 h treatment with respective CNCs, the cell culture medium was gently removed and 250 μL PBS, 2 μL of Calcein (40 μM) and 2 μL of ethidium homodimer-1 2 mM were added, mixed, and the cell suspension was incubated in the dark RT for 15 min. The loading solution was discarded and 250 μL of cold PBS + EDTA was added to each well and the plate was placed on ice for 30 min to facilitate the release of PBMCs from the plate. The bottom of the wells was scraped with a pipette tip and the cells were removed by gentle pipetting up/down, 100 μL of the cell suspension was utilized to assess live and dead cells by flow cytometry (BD Accuri™ C6 cytometer, BD, Franklin Lake, NJ, USA) using FL-1 filter Calcein (green, live) and FL-2 filter Ethidium homodimer (red, dead). The percentage of labeled cells was calculated by BD Accuri software using H2O2 treated cells as positive control for gating dead cells. The calculated data was expressed using the average of fold of change in the (%) of dead cells (ethidium bromide staining) corrected by (%) dead cells in the control (non-treated cells), from at least 4 experiments.
