The studies were approved by the Northwestern University Institutional Review Board, and all donors gave informed written consent. RA and normal synovial tissue fibroblasts were isolated from fresh synovial tissues by mincing and digesting in a solutionof dispase, collagenase, and DNase (20 (link), 21 (link)). Cells were used between passages 3–9. RA and normal synovial tissue fibroblasts were treated with IL-17 for 0–8h for mRNA studies and cell supernatants were harvested after 24h for protein studies. Monocytes were separated from buffy coats (Lifesource, Chicago, IL) obtained from healthy donors. Mononuclear cells, isolated by Histopaque (Sigma-Aldrich, St. Louis, MO) gradient centrifugation, were separated by countercurrent centrifugal elutriation. Monocytes were used for chemotaxis or allowed to differentiate to macrophages as previously described (22 (link), 23 (link)). Macrophages were treated for 0–8h for mRNA studies and 24h for protein studies.
Protocol detail
Isolation and Culture of RA and Normal Synovial Fibroblasts
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Cells
Centrifugation
Chemotaxis
Collagenase
Countercurrent
Dispase
Dnase
Donors
Fibroblasts
Histopaque
Il 17
Institutional review board
Macrophages
Monocytes
Mrna
Normal tissue
Protein
Synovial tissue
Corresponding Organization :
Other organizations : Louisiana State University Health Sciences Center New Orleans, Jesse Brown VA Medical Center
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Variable analysis
independent variables
- IL-17 treatment duration (0-8 hours)
- MRNA expression (in RA and normal synovial tissue fibroblasts, and macrophages)
- Protein secretion (in RA and normal synovial tissue fibroblasts, and macrophages)
- Cell type (RA and normal synovial tissue fibroblasts, monocytes, and macrophages)
- Cell passage number (3-9)
- Duration of treatment (0-8 hours for mRNA studies, 24 hours for protein studies)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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