MSC were always harvested by trypsinisation upon 80% confluent growth, counted with a Neubauer counting chamber (Brand, Wertheim, Germany) and additionally with a CASY cell counter (Schärfe System, Reutlingen, Germany) and re-seeded in a density of 10,000 cells/cm2 in culture flasks (Nunc Thermo Fisher Scientific, Langenselbold, Germany). Different passages were compared as indicated in sample title.
Growth protocol
MSC from adipose tissue (MSC-AT) were isolated from lipoaspirates after written consent according to the guidelines of the Ethics Committee of the University of Aachen (Permit number 163/07). Lipoaspirates were washed in 9 g/L NaCl, digested with 2 g/L collagenase type I (PAA, Pasching, Austria) and passed through a 100 µm cell strainer as described before (Cholewa et al., 2011). Culture medium consisted of Dulbecco´s Modified Eagles Medium-Low Glucose (DMEM-LG; PAA) with 2 mM L-glutamine (Sigma Aldrich, St. Louis, MO, USA), 100 U/mL penicillin/streptomycin (pen/strep; Lonza, Basel, Switzerland) and 10% human platelet lysate (HPL) which was pooled from five platelet units of healthy donors as described before (Horn et al., 2010). Cells were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide with medium changes twice per week.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA of 10(6) MSC was isolated using the QIAGEN DNA Blood Midi-Kit. DNA quality was assessed with a NanoDrop ND-1000 spectrometer (NanoDrop Technologies, Wilmigton, Del) and average fragment length was assessed by gel electrophoresis.
Label
biotin
Label protocol
As per manufacturer (Affymetrix)
Hybridization protocol
DNA was restriction digested, PCR amplified, fragmented, labeled and hybridized to each array according to the manufacturer's instructions.
Scan protocol
The Arrays were then washed using Affymetrix fluidics stations, and scanned using the Gene Chip Scanner 3000 7G.
