Differential effects of TNFalpha and IL1beta on FLS global gene expression profile

Fibroblast-like cells (FLS) were purified from synovial biopsies from RA patients. Briefly, minced synovial fragments were digested in 1 mg/ml hyaluronidase solution (Sigma Aldrich, St Louis, MO) for 15 minutes at 37°C and 6 mg/ml collagenase type IV (Invitrogen, Paisley, UK) for 2 hours at 37°C. Next, cells were washed, resuspended in high-glucose Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 1% antibiotics-antimycotics (Invitrogen) and 1% MEM sodium pyruvate (Invitrogen), and seeded at 10,000 cells/cm2 in 6-well plates. When the cells reached confluence, adherent cells were detached using sterile 0.5% trypsin-EDTA (Invitrogen) and used as FLS between passages 3 and 9.Cells were seeded in 24-well plated at 25.000/well and incubated overnight with or without the following cytokines : TNF-α (R&D Systems, Minneapolis, MN) 10 ng/ml, IL-1β (R&D Systems) 10 ng/ml. After overnight incubation with the indicated cytokines, cells were harvested and total RNA was extracted using the Nucleospin® RNA II extraction kit (Macherey-Nagel, Düren, Germany), in order to be labeled according to a standard Affymetrix procedure and hybridized on HGU133 Plus 2.0 Human Genome slides.


Primary Purpose
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Description of Cohorts
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Informed Consent Given?
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Multi-center Study?
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Organisms
Homo sapiens
Number of subjects
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Diseases
rheumatoid arthritis
Sample Sources
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Type of Samples Collected
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Age Range of Study Participants
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BMI Range of Study Participants
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