Fig. 2
Plasma-treated medium (PTM) induces a cytotoxic effect and alterations in LLC cells. (a) Lung normal MRC5 cells, (b-c) tumor cells (LLC or 4T1 cells), and (d) primary cells (DC) were incubated with various concentrations of PTM (6.25, 12.5, 25, and 50%) for 24 h. Cell viability was determined using CCK-8 assays. This experiment was repeated thrice. Statistical significance was analyzed via one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the control. n.s., not significant. (e) On day 1, intracellular ATP in LLC or DCs were assayed using luciferase-based viability following treatment with 25% PTM alone or pre-incubation with 2 mM PYR, 10 mM NAC, and 0.1 mM cPTIO for 1 h before PTM treatment. The mean ± SEM represents data; *p < 0.05 and ***p < 0.001; two-way analysis of variance (ANOVA). (f) LLCs were incubated alone (25% or 50% PTM) or co-cultured with splenocytes (1 × 105/well of 96-well plates) in the presence of PTM. Cell viability was assessed using CCK-8 assays. (g-h) LLC and DC cell death incubated in various PTM was evaluated via Annexin/PI staining and flow cytometry, respectively. The positive sample inactivates heated at 100℃ for 10 min. Results are represented by the means ± SD from three independent experiments. Significant differences were determined via a two-way ANOVA; **p < 0.01 and ***p < 0.001 compared to the control