Iodonitrotetrazolium chloride

Cell viability was estimated by quantifying the release of LDH in the culture supernatant, as previously described (Mathy-Hartert et al., 2009 (link)). A sample of the supernatant or dilutions of the standard solution (LDH from rabbit muscle) was mixed with Tris buffer [10 mM Tris-HCl (pH 8.5) and, 0.1% bovine serum albumin] containing 800 mM lactate. Then, a colorimetric reagent containing 1.6 mg/ml iodonitrotetrazolium chloride (Sigma-Aldrich), 4 mg/ml nicotinamide adenine dinucleotide (Roche Diagnostics, Brussels, Belgium), and 0.4 mg/ml phenazine methosulfate (Sigma-Aldrich) was added, and the absorbance at 492 nm was read after 10 min of incubation at room temperature.

The cell culture media (DMEM, MEM, RPMI 1640), 2-mercaptoethanol (ME), horse serum, sodium pyruvate, and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific. Fetal calf serum (FCS) for Caco-2 and HT29-MTX-E12, non-essential amino acids (NEAA), L-glutamine, D-glucose, penicillin/streptomycin (P/S), trypsin, phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-γ), lipopolysaccharides (LPS), Accutase, β-nicotinamide adenine dinucleotide sodium salt (NAD), lithium L-lactate, phenazine methosulfate (PMS), iodonitrotetrazolium chloride (INT), Triton X-100, bovine serum albumin (BSA), and 50 nm amine-modified polystyrene nanobeads (PS-NH₂) were purchased from Sigma-Aldrich/Merck. Paraformaldehyde (PFA) was ordered from Carl Roth (Germany). The DQ12 quartz sample used in this study was kindly provided by the Institute of Occupational Medicine (IOM, Edinburgh, UK).

SUM225 and SUM159 cell lines and their derived cell lines were seeded in 6-well plates with a density of 5,000 cells/well in the presence of Blasticidin (10 μg/ml) (Invivogen, San Diego, CA) for 2–3 weeks. The same experimental setting was applied to both SFIHE and SFIH culture media. For analysis, cells were stained with crystal violet and the number of transformed foci was determined. To assess anchorage-independent growth, triplicate samples of 5 × 10⁴cells from each cell line were mixed 4:1 (v/v) with 2.0% agarose in growth medium to a final concentration of 0.3% agarose. The cell mixture was plated on top of a solidified layer of 0.5% agarose in growth medium (SFIHE). Cells were fed every 3 days with growth medium (SFIHE). Cells were stained with 0.02% iodonitrotetrazolium chloride (Sigma-Aldrich, St. Louis, MO) and photographed after 21 days. Colonies in the entire well were counted using a dissecting microscope and colonies larger than 50 μm were included. Crystal violet staining was used to count the colonies after the 21-day period. Experiments were performed in duplicate and repeated three times.