Fig. 1.

Gas plasma affected the viability, metabolic activity, and motility of dermal fibroblasts and regulated the molecular machinery of integrin adhesions. (a) Experimental workflow. Primary dermal fibroblasts (pFDs) were isolated from the skin of SKH1 mice and cultivated over ten days in fibroblast medium. pDFs were incubated with medium exposed to gas plasma for 20 s, 60 s, or 180 s, or were left untreated (ctrl), and analyzed by several down-stream assays. Representative phase-contrast images of the progression of pDFs after isolation from the skin of SKH1 mice on d2 and d7. Scale bars are 100 μm. F-actin stress fibers were visualized using FITC-phalloidin fluorescent probe (green); arrowheads show cell border staining in untreated (ctrl) and gas plasma-treated cells (pl-20 s). Scale bar is 50 μm (b) Live/dead assay measured at 6 h after gas plasma treatment, demonstrating mainly viable cells (green, calcein-positive) along with a few dead cells (red, propidium iodide-positive, pl-20 s) in comparison to control (ctrl). Scale bar is 50 μm. Quantification of metabolic activity of pDFs after 6 h, 24 h, and 72 h following gas plasma treatment. (c) Representative images showing migration of pDFs in untreated and gas plasma-treated (pl-20 s) pDFs. Scale bar is 200 μm. Quantification of wound closure and migratory behavior of pDFs up to 48 h after gas plasma treatment measured by wound healing scratch assay. (d) pDFs were harvested and lysed at 0.5 h, 2 h, 6 h, and 24 h after gas plasma treatment (20 s). qPCR of syndecans (SDC1/4), integrins (ITGA5, ITGB1), small GTP binding proteins (RAC, RHOA), tensin 1 (TNS), and fibronectin 1 (FN1) was perfomed in pDFs. Statistical analysis was done by unpaired two-tailed Student's t-test with significances of *p < 0.05, **p < 0.01, and ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)