A non-thermal atmospheric pressure plasma jet kinpen (neoplas GmbH, Greifswald, Germany) ionized a flow of argon gas. A voltage of 2-6 kVpp was applied with a frequency of around 1 MHz. Gas flow was set to 3 sLm (standard liters per minute) which was controlled by a mass flow controller (MKS Instruments, Germany). The treatment time was to 20 s, 60 s, and 180 s corresponding to the following treatment time equivalents: 100, 300, and 900 ms*µL/cell, respectively after plasma treatment of 5 ml of RPMI medium were plasma-treated and immediately transferred to 1×106 cells (indirect treatment). Two hour after exposure to treated medium, cells were collected, centrifuged, and washed. Alternatively, cells were incubated with fresh media for another 1 h, 4 h, or 22 h. Cells treated with 100 µM H2O2 served as positive controls for oxidative stress. To exclude effects of the carrier gas, cells incubated with argon gas treated medium were used as negative controls. Subsequently, all samples were analyzed and compared in respect to their gene activity.
Growth protocol
The keratinocyte cell line (HaCaT), a human adult (low calcium, high temperature) epithelial keratinocyte cell line, was seeded at a density of one million cells in 6 cm dishes in RPMI (Roswell Park Memorial Institute 1640 cell culture media) supplemented with 8 % fetal calf serum (Sigma), 0.1 mg/mL penicillin/streptomycin and 2 mM L-glutamine (Lonza, Basel, Switzerland) and were left to attach for 24 h prior to plasma treatment.
Extracted molecule
total RNA
Extraction protocol
Total RNAs from each group (n>6) were purified using Total RNA Mini Kit (Bio&Sell, Germany) according to instructions including DNaseI treatment (Qiagen, Germany). RNA integrity was confirmed using the Bioanalyzer2010 (Agilent, Germany). cDNA was synthesized by SuperScript double-stranded cDNA Synthesis Kit (Invitrogen, Germany) from 10 µg of total RNA using oligodT primer (200 ng/mL). Double strand cDNAs were end-labeled with fluorescent Cy3-dye using a One-Color DNA Labeling Kit (Roche NimbleGen, Germany).
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc. MS 200, Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
HaCaT untreated control
Data processing
The raw data (.pair file) were subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction was performed as implemented in the NimbleScan software package (Roche NimbleGen, Inc.).
