6 well clear multiwell plates

We evaluated In₂O₃-induced cytotoxicity by trypan blue exclusion assay. RAW 264.7 mouse macrophage cells (5 × 10⁵ cells/ml, RIKEN BioResource Center, Tsukuba, Japan) were seeded in 1.2 ml of DMEM containing 5% (v/v) FBS and 100 mg/l kanamycin in 6 Well Clear Multiwell Plates (BD Falcon, Franklin Lakes, NJ, USA). Immediately after seeding, the cells were incubated with 0-50 μg/ml of In₂O₃ for 24 h at 37°C in an atmosphere containing 5% CO₂. We employed these concentrations of In₂O₃, because we have previously demonstrated that other types of nanomaterials induced significant cytotoxic and/or genotoxic effects at similar concentrations^{18 (link),20) (link)}. Then, the cell suspensions were mixed with trypan blue, and the viability was calculated with a TC20 Automated cell counter (Bio-Rad Laboratories, Hercules CA, USA).

To analyze NO released from In₂O₃-treated cells, we measured the concentration of its products, nitrate (NO₃^-) and nitrite (NO₂^-), in the culture supernatant by using the Griess method. RAW 264.7 cells (5 × 10⁵ cells/ml) were seeded in 1.2 ml of phenol red-free DMEM (Gibco/BRL) containing 5% (v/v) FBS and 100 mg/l kanamycin in 6 Well Clear Multiwell Plates (BD Falcon). Then the cells were treated with 20 μg/mlIn₂O₃ for 2 or 4 h at 37°C. The culture supernatant was collected and centrifuged at 40,000 × g for 10 min at 4°C to remove In₂O₃ particles. To reduce NO₃^- to NO₂^-, the supernatant was incubated with 0.1 units/ml of nitrate reductase from Aspergillus niger (Sigma-Aldrich) in the presence of 1 mM glucose-6-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 0.3 units/ml of glucose-6-phosphate dehydrogenase and 20 μM NADPH (Oriental Yeast, Tokyo, Japan) for 30 min at room temperature. The reaction mixture was incubated with 0.25% (w/v) sulfanilamide (Griess reagent I, Wako) and 0.025% (w/v) naphthylethylenediamine (Griess reagent II, Sigma-Aldrich) in 0.625% (v/v) phosphoric acid for 10 min at room temperature. The absorbance was measured at 540 nm with a microplate reader (Model 680, Bio-Rad laboratories) and NO₂^- concentration was determined by comparison with a standard curve generated with sodium nitrate (NaNO₂, Wako).