cell line: HaCaTs (DKFZ), a human adult (low calcium, high temperature) epithelial keratinocyte cell line treatment: H2O2 (positive control)
Treatment protocol
Non-thermal plasma was generated using the experimental setup schematically illustrated in Figure 1. An 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 efficiency of 300 ms*µL/cell (treatment time equivalents) corresponded to 60 s treatment time in 5 mL RPMI per 1×106 cells (unpublished data). Immediately after RPMI plasma treatment medium was transferred to the cells (indirect treatment). One hour after exposure to treated medium cells were collected, centrifuged, and washed. Alternatively, cells were incubated with fresh media for another 2 h, 5 h, or 23 h. Cells treated with 50 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 HaCaTs cell line (DKFZ), a human adult (low calcium, high temperature) epithelial keratinocyte cell line, was seeded at a density of 1×106 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>8) were purified using Total RNA 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 (200 ng/mL) and random hexamer primer (100 ng/mL). Double strand cDNA were end-labeled with fluorescent Cy3 dye using a One-Color DNA Labeling Kit (Roche NimbleGen, Germany).
Label
Cy3
Label protocol
abeling 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 treated positive control
Data processing
The raw data (.pair file) was 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 as implemented in the NimbleScan software package (Roche NimbleGen, Inc.).
