Adipose tissue was harvested using a tumescent technique with pump-assisted aspiration (Oedayrajsingh-Varma MJ et al, 2006). Adipose tissue derived stromal cells (ASCs) were isolated as previously described (Zuk PA et al, 2001) with slight modifications. In brief, prior to digestion, the tissue was washed three times in equal volumes of prewarmed D-PBS and then digested in equal volumes of collagenase buffer, 0.28Wünch U/ml crude collagenase mix (Lot. No. LTQ5230; Wako, Neuss, Germany) in D-PBS with 20mg/ml BSA (Roche Applied Science, Hvidovre, Denmark). Following digestion at 37°C with gentle agitation for 1 hour, the released cells were isolated by fractional sedimentation centrifugation at 400 x g for 10 min and the pelleted cells were filtered through a 70 μm mesh cell strainer (BD Bioscience, Broendby, Denmark). Contaminating erythrocytes were lysed using erythrocyte lysis buffer (8,3g/l NH4Cl (Merck Chemicals, Darmstadt, Germany) and 0.993g/l K2HPO4 (Bie & Berntsen, Aabyhoej, Denmark) in 0.04g/l EDTA-Na (Merck). The remaining nucleated cells were further purified through a second round of centrifugation and filtration and seeded at a cell density corresponding to 150μl adipose tissue/cm2 in growth medium. Twelve hours post seeding the medium was changed to remove nonadherent cells. The freshly isolated cells were expanded for 11 days before cryopreservation and storage at -140°C. The ASCs were thawed and cultured for one week in growth medium in a humidified atmosphere containing 5% CO2 buffered with ambient air at 37°C. At 80% confluence the cultures were passaged at 1000 cells/cm2 and left for 24 hours to adhere.
Growth protocol
The ASC cultures were maintained in growth medium, alpha Modified Eagle Medium (αMEM, Invitrogen, CarlsBad, Ca) supplemented with 1% Penicilin/Streptamicin (Invitrogen), 0.5% Gentamicin (Invitrogen) and 10% Fetal Calf Serum (Invitrogen) in a humidified atmosphere, containing 5% CO2 buffered with air of varying oxygen tensions (ambient, 15, 10, 5 and 1% oxygen tension) at 37°C and with medium change twice a week.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cultured adipose tissue derived stem cells using the BioRad Aurum total RNA mini kit (Bio-Rad, Copenhagen, Denmark) according to manufacturer's instructions.
Label
Biotin
Label protocol
With 400 ng totalRNA as entry material, the Illumina Total Prep RNA Amplification Kit (Illumina Inc, San Diego, CA) was used according to standard protocols to produce in vitro transcribed and biotin labeled cRNA.
Hybridization protocol
Biotin labled cRNA (1.5μg) was hybridized to Illumina Human-6 v2.0 arrays using the IlluminaHybChamber and gasket for 16 hours at 58ºC. After a series of washes, including high temperature (55ºC) wash, room temperature washes and an EtOH wash, the arrays were blocked for 10min and developed using streptavidin-Cy3. All buffers, blocking and staining solutions were from llumina and were used according to standard protocol (Whole-Genome Gene Expression with IntelliHybTM Seal Experienced User Card, Illumina Part # 11226048, Rev B.). Before scanning the arrays were dried by centrifugation at 275RCF for 4min.
Scan protocol
Illumina Human-6 v2.0 arrays were scanned on the Illumina Bead station 500GX System
Description
One outlier sample was excluded from the normalisation step (Ambient 0days Sample A). 1842761028_B
Data processing
Raw intensity values were background subtracted using Illumina's BeadStudio software version 3.0.14 and exported into the R Bioconductor statistical package where the data were transformed and normalised using the vsn2 BioConductor package (Huber et al., 2002).
