E epidermis was separated from tissues utilizing sterile forceps, and after that digested with 0.125 trypsin (Gibco, Life Technologies, Carlsbad, CA, USA) for 15 min at 37 . The samples had been treated with KGMGoldTM keratinocyte growth medium Carboxypeptidase D Proteins Gene ID containing supplements (Lonza Bioscience, Basel, Switzerland) to inactivate the trypsin. Soon after filtering and centrifugation at 12009g for 3 min, the pellet was suspended in keratinocyte culture media and cultured at 37 in a humidified atmosphere containing 5 CO2. Cells in the second passage were applied to subsequent experiments. 2.2 LTP device The LTP method was as utilised in our prior study . The LTP was a created dielectric barrier discharge device, and applied 13 kHz of frequency, 5.9 kV of voltage, and 42 W of electric power. The functioning gas for LTPTissue Eng Regen Med (2019) 16(6):585generation was a mixture of 5000 ccm of helium and 50 ccm of air at 28 2 . two.3 LTP therapy Keratinocytes have been seeded in 35-mm cell culture dishes at a density of five 9 104 cells, and cultured for 2 days. Culture dishes had been washed once with Dulbecco’s phosphate buffered saline (DPBS) and added to 1.2 ml of DPBS just before LTP remedy. Untreated dishes have been subjected towards the Protein tyrosine phosphatases Proteins Purity & Documentation similar method. The distance involving the LTP torch and culture dish was 3 cm plus the treatment diameter was two cm. The cells had been treated with LTP for 30 s, 1 min, or three min, based on the experiment. Analyses have been performed six and/or 24 h immediately after LTP remedy. two.4 Cell viability assay Keratinocyte viability was measured by an improved MTT assay strategy (EZ-Cytox, Dogen, Seoul, Korea) in line with the manufacturer’s guidelines. The final worth was calculated in accordance with the following formulae: sample absorbance – background absorbance = original absorbance; original absorbance/control absorbance 9 100 = viability. two.five Cell migration assay Keratinocyte migration was measured by wound healing assays in 35 mm l-dishes with 2-well culture inserts (Ibidi GmbH, Planegg, Germany) in accordance with the manufacturer’s guidelines. Keratinocytes were plated in the culture insert dish at a density of 2 9 104 cells per effectively and cultured for 24 h. The culture insert was then withdrawn, which made a defined cell-free wound of 500 50 lm. Mitomycin C (Sigma, St. Louis, MO, USA) was added at five lg/ml towards the cell culture medium to inhibit cell proliferation for the duration of migration. The images of cell migration within the wound region have been captured 6 and 24 h right after exposure to LTP for 30 s or three min employing a light microscope (IX 70, Olympus, Tokyo, Japan). The migration was normalized to that of untreated keratinocytes as a manage, which was set to 100 , and expressed as a fold-change. two.6 Cytokine array The supernatants of keratinocyte cultures were collected 24 h after exposure to LTP for 1 or three min. The supernatants from untreated cells had been made use of as controls. Cytokine levels had been measured by cytokine arrays (R D, Minneapolis, MN, USA), which integrated nine cytokine targets, especially GM-CSF, IL-1b, IL-4, IL-6, IL-8, IL10, IL-17, IL-12, and IL-13.two.7 Enzyme-linked immunosorbent assay The supernatants of keratinocyte cultures had been collected 24 h soon after exposure to LTP for 30 s or 3 min, or from untreated manage cells, and have been analyzed by enzymelinked immunosorbent assay (ELISA) (Cusabio Technology, Wuhan, China) for eight selected molecular targets as follows: PDGF-A, PDGF-B, VEGF-A, HB-EGF, vascular angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), fibroblast development factor.