Lipoaspirates (100-200g per aspirate) were obtained from subcutaneous abdominal adipose of women undergoing elective liposuction. Lipoaspirate was repeatedly washed with PBS until blood was completely removed from the tissue, and then incubated with equal volume of DMEM containing collagenase (0.1 %, Sigma Aldrich) for 30 min at 37°C in a shaking incubator at 110 rpm, followed by incubation in 4°C, while still in collagenase and nutritionally deficient medium (no FCS), for 16 hours under severe hypoxia conditions. Digested material was then centrifuged at 1500 rpm for 10 minutes at 4°C. Supernatant containing adipose cell debris (dead adipocytes, macrophages, red blood cells, adipose stem cells among other cell components) was removed by aspiration and the remaining cell pellets were washed several times with PBS. Pellets were resuspended in PBS and incubated with a red blood cell lysis buffer (eBiosciences, San Diego, CA) for 10 min at R/T (2X). Remaining cell pellets contain cells highly resistant to severe cellular stress, and these were resuspended in Dulbecco’s Modified Eagle Medium 1x (DMEM; CellGro, MediatechInc, Manassas, VA) containing 10% fetal bovine serum (FBS; Thermo Scientific Hyclone, Logan, UT) and 5% antibiotic-antimyocotic solution (CellGro, Mediatech Inc, Manassas, VA) and plated as cells in suspension as well as adherent cells. For ASC isolation, lipoaspirate material was subjected to collagenase digestion (0.1 %, Sigma Aldrich) for 30 min at 37°C in a shaking incubator at 110 rpm, and ASCs were isolated and cultured as previously as previously described (Zuk, et al. Mol Biol Cell. 2002 December; 13(12): 4279–4295).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using an RNeasy Mini Kit (Qiagen).
Label
Cy3
Label protocol
Agilent standard protocol
Hybridization protocol
Agilent standard protocol
Scan protocol
Chips were scanned using the Agilent Technologies G2505C Microarray Scanner System
Description
ASC_rep3
Data processing
gProcessedSignal(normalized) columns were further processed using GeneSpringGX version 12.1.0 (Agilent).
