Kaluza 2.1.3 analysis software

Viable single-cell suspensions of tumors were retrieved using the GentleMacs tumor dissociation kit and the OctaMacs device (both Miltenyi BioTec, Mönchengladbach, Germany) according to the supplier’s instructions. The remaining erythrocytes were lysed using red blood cell lysis buffer (BioLegend, Amsterdam, The Netherlands). After washing, cells were stained with antibodies targeting (conjugate) IA/IE (APC-Fire 750), CD80/86 (APC), CD45 (AF700), F4/80 (PerCp-Cy5.5), CD3 (BV421), CD8 (BV510), CD11c (BV605), CD4 (BV785; all BioLegend, Amsterdam, the Netherlands), MHCI (BUV661; both BD Biosciences, Heidelberg, Germany), and iFluor maleimide 860 (Biomol, Hamburg, Germany) for live–dead discrimination for 30 min at 37 °C. After washing, cells were acquired using flow cytometry (CytoFLEX LX; Beckman-Coulter, Krefeld, Germany) and analyzed using Kaluza 2.1.3 analysis software (Beckman-Coulter, Krefeld, Germany).

For the discrimination between viable and dead cells, 4’6-diamidino-2-phenylindole (DAPI, 1 µM; Sigma-Aldrich, St. Louis, MO, USA) and CellEvent Caspase-3/7 Green Detection Reagent (1 µM; Thermo Fisher Scientific, Waltham, NJ, USA) were utilized. After 15 min of incubation at 37 °C, the percentage of each cell population staining negative or single- or double-positive was determined using flow cytometry (CytoFLEX S; Beckman-Coulter, Krefeld, Germany) and Kaluza 2.1.3 analysis software (Beckman-Coulter, Krefeld, Germany).

Cell cycle analysis and assessment of intracellular ROS levels was performed on fixed and permeabilized cells 24 h after gas plasma treatment. Nucleic staining was done using 10 µM 4′,6 diamidine-2-phenylindole (DAPI; Sigma‑Aldrich, Germany), and a PE-conjugated antibody was utilised to stain 3-nitrotyrosine (Santa Cruz Biotechnology, USA; Cat# sc-32757 PE) for 20 min at 37 °C. After washing, cells were acquired using flow cytometry (CytoFLEX LX; Beckman-Coulter) and evaluated using Kaluza 2.1.3 analysis software (Beckman-Coulter).