high metastatic potential; growth under reduced oxygen
Treatment protocol
Two human pancreatic carcinoma cell lines: FG, L3.6pl (Bruns et al., 1999) were used in this study. Both cell lines were cultivated in Dulbecco's Modified Eagle Medium (Gibco/Invitrogen) supplemented with 10% foetal bovine serum (Biochrom, Lot No.: 923 FF), 1x Glutamax-1 (Gibco/Invitrogen), 2x MEM vitamin solution (PAN Biotech, Aidenbach Germany), 2x MEM NEAA (PAN Biotech, Aidenbach Germany), 1x Penicillin/streptomycin (100 U/ml Penicillin + 0,1 mg/ml streptomycin) (PAN Biotech, Aidenbach Germany). Cells were cultivated at 37°C and 5% CO2 starting from frozen stock without further passaging. 500000 frozen cells were plated into 175cm2 Nunclon Δ-treated flask with filter cap (Nunc Roskilde Denmark) containing 25 ml medium and cultivated until an apparent confluence of 80% was reached (typically 9 days). Medium was changed every second day. The last 24 hours cells were treated by hypoxia where air was replaced by nitrogen to reach an oxygen concentration of 1% or left untreated respectively.
Growth protocol
Two human pancreatic carcinoma cell lines: FG, L3.6pl (Bruns et al., 1999) were used in this study. Both cell lines were cultivated in Dulbecco's Modified Eagle Medium (Gibco/Invitrogen) supplemented with 10% foetal bovine serum (Biochrom, Lot No.: 923 FF), 1x Glutamax-1 (Gibco/Invitrogen), 2x MEM vitamin solution (PAN Biotech, Aidenbach Germany), 2x MEM NEAA (PAN Biotech, Aidenbach Germany), 1x Penicillin/streptomycin (100 U/ml Penicillin + 0,1 mg/ml streptomycin) (PAN Biotech, Aidenbach Germany). Cells were cultivated at 37°C and 5% CO2 starting from frozen stock without further passaging. 500000 frozen cells were plated into 175cm2 Nunclon Δ-treated flask with filter cap (Nunc Roskilde Denmark) containing 25 ml medium and cultivated until an apparent confluence of 80% was reached (typically 9 days). Medium was changed every second day. The last 24 hours cells were treated by hypoxia where air was replaced by nitrogen to reach an oxygen concentration of 1% or left untreated respectively.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cultured cells with the Trizol method according to the manufacturers protocol
Label
biotin
Label protocol
total RNA was transcribed and labeled with Ambion Kit MessageAmp II Biotin Enhanced according to the manufacturers protocol
Hybridization protocol
fragmented cRNA was hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays and washed and stained with Affymetrix Wash&Stain Kit on an Affymetrix Fluidics Station 450
