Thermo mutliscanmk3

Cells were seeded in 96‐well culture plates, and cell proliferation was determined after incubation for 24 and 48 hours using a cell counting kit‐8 (CCK8; Dojindo, Japan). Growth curves were generated from a colorimetric assay. The absorbance value of each well at 450 nm was measured with a micro‐plate reader (Thermo MutliscanMK3; Thermo Fisher Scientific, Waltham, USA).

Total NO concentration in culture medium and cells was calculated by measuring the nitrate and nitrite concentrations with a Total Nitric Oxide Assay Kit (Beyotime, China) according to the manufacturer’s instructions. The optical densities at 540 nm were recorded using a Microplate Reader (Thermo MutliscanMK3; Thermo Fisher Scientific, Waltham, MA, USA). The concentration of NO output was calculated from the standard curve. Urea production was determined using a Urea Colorimetric Assay Kit (BioVision). Five million macrophages were harvested and lysed for 30 min in 100 μL of 10 mM Tris–HCl, pH 7.4, containing 0.4% (w/v) Triton X-100. Then, the cells were centrifuged at 13,000 rpm for 10 min. The supernatant was collected for the Arginase Activity Assay kit (Sigma, MAK112). The quantitative data were expressed as the mean ± SD. One-way ANOVA was used to determine the statistical significance.

Cell viability was detected by the Cell Counting Kit-8 (CCK-8) assay, which was conducted according to the manufacturer’s instructions. Briefly, OSCC cells (Cal-27 and Scc-9) were seeded in 96-well culture plates with the different treatments as indicated. The ANE or cisplatin was added to the cell culture at varying concentrations after cell seeding, and cell proliferation was determined after incubation for 24, 48, and 72 h. The cells were then incubated with 10 μL of CCK-8 and 100 μL of DMEM at 37 °C for another 2 h, after which the absorbance at 450 nm was measured with a micro-plate reader (Thermo MutliscanMK3; Thermo Fisher Scientific, Waltham, MA, USA). Cell viability curves were generated using a colorimetric assay.