|
| Status |
Public on Sep 20, 2008 |
| Title |
Chondrocytes stimulated with methotrexate treated RASF rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Human chondrocytes isolated from lateral condyle of femur bone in culture from donor pool 11, stimulated with supernatant from methotrexate treated RASF
|
| Organism |
Homo sapiens |
| Characteristics |
Human chondrocytes cultured in RPMI 1640 medium supplemented with 10% human serum, 100ng/ml amphotericin B, 100U/ml penicillin and 100µg/ml streptomycin for 2 passages, 3D cultured for 14 days in alginate beads in presence of 170µM L-ascorbic acid 2-phosphate and subsequently stimulated for 48h with supernatant of RASF cultured in the same medium and treated with methotrexate for 48h
|
| Treatment protocol |
Human chondrocytes were isolated by enzymatic digestion (1 U collagenase P, 333 U collagenase II and 33 U hyaluronidase), passage 2 chondrocytes were encapsulated in alginate beads (20 Mio cells/ml in 1,5% (w/v) alginate) and stimulated with conditioned medium of NDSF, RASF or treated RASF
|
| Growth protocol |
Human chondrocytes obtained from lateral condyle of femur bones of 6 donors post mortem were amplified by cultivation in RPMI 1640 medium supplemented with 10% human serum, 100ng/ml amphotericin B, 100U/ml penicillin and 100µg/ml streptomycin for 2 passages, subsequently 3D cultured in alginate beads for 14 days in presence of 170µM L-ascorbic acid 2-phosphate and finally stimulated with conditioned medium of RASF, NDSF, or treated RASF
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Prior to RNA extraction, alginate beads were solubilized on ice in 55mM sodium citrate, 30mM EDTA and 150mM NaCl and cells were centrifuged at 800g for 5min (4°C). Total RNA isolation was proceeded according to the manufacturer’s protocol. Additionally, a proteinase K and DNase I digestion were performed. Isolation of total RNA was done for the different stimulated donor chondrocytes separately. Afterwards, equal amounts of total RNA from three stimulated donor chondrocytes were pooled, yielding different experimental groups of chondrocytes stimulated with supernatant of NDSF, RASF or treated RASF
|
| Label |
biotin
|
| Label protocol |
2.5 µg of total RNA was amplified and labeled using the GeneChip® one-cycle target labeling and control reagents (Affymetrix)
|
| |
|
| Hybridization protocol |
50µg/ml of labeled cRNA was hybridized at 45°C for 16h using the Affymetrix GeneChip hybridization oven. Washing and staining was performed on the Affymetrix Fluidics 450 using the EukGE-WS2v4 protocol.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000
|
| Description |
Gene expression data of human chondrocytes stimulated with supernatant of RASF treated for 48h with methotrexate
|
| Data processing |
Data were processed with GCOS 1.4 (TGT = 150) and subsequently imported and further analyzed in the BioRetis online database (www.bioretis.de). Additionally, expression data was processed with Robust Multi-Array Analysis version 0.4a7 (RMA)
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| |
|
| Submission date |
Sep 19, 2008 |
| Last update date |
Sep 19, 2008 |
| Contact name |
Thomas Häupl |
| E-mail(s) |
thomas.haeupl@charite.de
|
| Phone |
+49 30 450513293
|
| Organization name |
Charité
|
| Department |
Rheumatology
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE12860 |
Antirheumatic Drug Response in Human Chondrocytes: Potential Molecular Targets to Stimulate Cartilage Regeneration |
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