Tibiae, femurs and humeri were isolated from E15.5 mouse embryos and placed in α-MEM media (Invitrogen) containing 0.2% Bovine Serum Albumin (BSA), 1 mM β-glycerophosphate, 0.05 mg/ml ascorbic acid and penicillin/streptomycin incubated at 37°C in a humidified 5% CO2 incubator overnight. The following morning media was removed and the bones placed in 4 ml of 0.25% trypsin-EDTA (Invitrogen) for 15 min at 37oC. Trypsin was subsequently replaced with 1 mg/ml collagenase P (Roche) in DMEM / 10% fetal bovine serum (Invitrogen), and cells were incubated at 37°C with rotation at 100 rpm for 90 min. Following digestion, the cell suspension was centrifuged for 5 min at 1000 rpm, and the collagenase containing supernatant was decanted. Chondrocytes were resuspended in media containing 2:3 DMEM:F12, 10% fetal bovine serum, 0.5 mM L-glutamine, and penicillin/streptomycin (25 units/ml). Cells were seeded in 6-well NUNC plates at a density of 2.5X104 cells per ml and incubated overnight. Primary monolayer chondrocytes were treated with 100 µM TSA reconstituted in DMSO and diluted in fresh media supplemented with 0.25 mM ascorbic acid (Sigma) and 1 mM β-glycerophosphate (Sigma) and incubated for up to 24 hrs.
Biomaterial provider
SB90-treated embryonic primary chondrocyte cultures, incubated for 24hrs.
Treatment protocol
SEE CHARACTERISTICS
Growth protocol
SEE CHARACTERISTICS
Extracted molecule
total RNA
Extraction protocol
Qiagen RNeasy extraction
Label
SAPE
Label protocol
Completed according to the London Regional Genomics Centre (LRGC): http://www.lrgc.ca/
Hybridization protocol
LRGC specifications
Scan protocol
M.A.S. 5.0-see LRGC specifications
Description
AFFY543
Data processing
Microarray data were pre-processed RMAEXPRESS software v.0.4.1 developed by B. Bolstad, University of California, Berkeley. Background adjustment and quantile
normalization parameters were selected for data processing. Logarithmically transformed expression values were used to implement Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005, PNAS).
