hMSCs were plated at 1×105 cells/cm2 in complete culture medium and incubated at 37°C under hypoxia (0.5% O2, 5% CO2) with supplemental nitrogen or normoxia (21% O2, 5% CO2) for 24 hours using a well characterized, finely controlled ProOx-C-chamber system (Biospherix, Redfield, NY).
Growth protocol
Human bone marrow was extracted from healthy donors with no known pathologic condition after informed consent was obtained. After washed with phosphate-buffered saline (PBS) twice, total bone marrow were plated and cultivated in Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG; Invitrogen, Carlsbad, CA, USA) supplemented with 20% FBS (Invitrogen). After 48 hours culture at 37°C and 5% CO2, non adherent cells were removed, and medium was replaced every other day. Once confluent, adherent cells were further passaged and replated into new culture dishes. After 3-5 passages, these cells were resuspended as 5×105 cells/100 μl in PBS containing 1% bovine serum albumin for each antibody, and incubated with human specific monoclonal antibodies CD29-phycoerythrin (PE) (eBioscience), CD34-PE (MACS, Miltenyi Biotec), CD105-PE, CD117-PE and CD166-PE (all from BD Biosciences Pharmingen)， respectively. After 45 min of incubation at 4oC, cells were washed twice in PBS and at least 10,000 events were acquired for each sample using using BD FACSCantoTM II Flow Cytometry System.
Extracted molecule
total RNA
Extraction protocol
Total RNA from different cultured cells was extracted using Dr.P Kit (Biochain, Hayward, CA, USA), according to the manufacturer’s instructions.
Label
Cy3
Label protocol
1μg of total RNA was used to synthesize the double strand cDNA. RNA was amplified by in vitro transcription using Ambion’s MessageAmp™ II aRNA Amplification Kits (Life Technologies, Austin, TX, USA). Then, aRNA was reverse transcribed into cDNA and further labeled with cy3-dCTP with Klenow enzyme.
Hybridization protocol
According to the instructions of Agilent Gene Expression Hybridization Kit
Scan protocol
Scanned on an Agilent G2505C scanner. Images were quantified using Agilent Feature Extraction Software (version 10.7.3.1).
Description
Biological replicate 1 of 3,21% O2,subject1
Data processing
Agilent Feature Extraction Software (version 10.7.3.1) was used for background subtraction and LOWESS normalization.
