Casyton

Proliferation of cells was examined by cell counting (CASY Cell Analyzer, Roche Applied Science, Mannheim, Germany). Adherent cells, detached by trypsin/ethylenediaminetetraacetic acid (EDTA) treatment, were suspended in CASYton (Roche Applied Science) as 1 : 100 dilution. Measurement was performed using a capillary of 150 μm in diameter and cell line-specific gate settings to discriminate between living cells, dead cells, and cellular debris (Caki-1: 8.57 μm/15.4 μm, 786-O: 6.9 μm/14.7 μm, RCC4: 8.5 μm/15.75 μm, A-498: 7.2 μm/15.75 μm, and RC-124: 6.5 μm/12.75 μm).

Cell growth was determined after 4, 24, 48, 72, 96, and 120 h using a CASY cell counter and analyzer model TT (Roche Applied Science, Mannheim, Germany) with a 150 µm capillary. For this purpose, 1 × 10⁴ endothelial cells were suspended in 200 µL culture media and treated with CAP or carrier gas argon (control group) for 10 s (kINPen) or 30 s (Mini Jet). After treatment, the cell suspension was transferred to another 24-Well cell culture plate. The treatment well was rinsed with 200 µL fresh media, which was also transferred to the culture plate. Then, 800 µL fresh media was added, and cells were incubated in a humidified atmosphere at 5% CO₂ and 37 °C. Cell count was determined by suspending the cells by trypsin/EDTA (Ethylenediaminetetraacetic acid) treatment and diluting 100 µL cell suspension in 10,000 µL CASYton (Roche Applied Science, Mannheim, Germany). The measurement was performed three times with 400 µL each of this dilution and was performed in triplicate. To discriminate between cell debris, dead cells, and living cells, gates of 5.88 µm/11.13 µm were used.

Cell growth was determined after 4, 24, 48, 72, 96, and 120 h using a CASY cell counter and analyzer model TT (Roche Applied Science, Mannheim, Germany) with a 150 μm capillary. For this purpose, 1 × 10⁴ cells (SW1353) or 2 × 10⁴ cells (CAL-78) were suspended in 200 μL culture media and treated with CAP or carrier gas argon (control group) for 10, 30, and 60 s. The atmospheric plasma jet kINPen MED was used for CAP treatment. After treatment, 800 μL fresh media were added, and cells were incubated in a humidified atmosphere at 5% CO₂ and 37 °C. Cell count was determined by suspending the cells by trypsin/EDTA (Ethylenediaminetetraacetic acid) treatment and diluting 100 μL cell suspension in 10,000 μL CASYton (Roche Applied Science). The measurement was performed three times with 400 μL each of this dilution and was performed in triplicate. To discriminate against cell debris, dead cells, and living cells, gates of 7.20 μm/13.95 μm (SW1353) and 7.20 μm/14.85 μm (CAL-78) were used.

Aliquots of erythrocyte suspensions were diluted 10 or 40 times in PBS. For cell counting and quality control, samples were further diluted 1000 times in CASYton (Roche Applied Science, Penzberg, Germany) and their absolute number, diameter and volume determined using a CASY^TM cell counter (Roche Applied Science) employing a 60 μl capillary. Samples with aberrant peak shape or cell volume were excluded from further analyses. All measurements were performed in duplicates.