Cytokine-Induced Id1 Expression in Synovial Fibroblasts

HMVECs, EPCs, monocytes, and RA, OA, and NL synovial fibroblasts were plated in 6-well plates at 200,000 cells/well and serum starved overnight. Media was exchanged and collected after 24 h. The collected media was analyzed by ELISA for Id1 (MyBioSource, San Diego, CA, USA). Manufacturer protocols were followed. SFs of RA, OA, and several other diseases were also analyzed by this ELISA. Both synovial and dermal fibroblasts were stimulated with varying cytokines and concentrations. Cytokines used were tumor necrosis factor alpha (TNF-α, Thermo Fisher Scientific), chemokine (C-X-C motif) ligand 16 (CXCL16, R&D Systems, Minneapolis, MN, USA), interleukin 17 (IL-17, R&D Systems), and TGF-β (R&D Systems). These cytokines were chosen because they are known to be upregulated in RA ST [23 (link), 24 (link)]. The supernatant and exosome fractions were collected after 24 h and analyzed by this ELISA.

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Edhayan G., Ohara R.A., Stinson W.A., Amin M.A., Isozaki T., Ha C.M., Haines GK I.I.I., Morgan R., Campbell P.L., Arbab A.S., Friday S.C., Fox D.A, & Ruth J.H. (2016). Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis. Arthritis Research & Therapy, 18, 87.

Publication 2016

TNF-α CXCL16 IL-17 TGF-β

Cells Chemokine Cytokines Dermal Elisa Epcs Exosome Fibroblasts Il 17 Interleukin 17 Ligand Monocytes Serum Tgf β Tnf α

Corresponding Organization :

Other organizations : University of Michigan–Ann Arbor, Mount Sinai Health System, Henry Ford Hospital

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Variable analysis

independent variables

Cell type (HMVECs, EPCs, monocytes, RA, OA, and NL synovial fibroblasts)
Cytokine treatment (TNF-α, CXCL16, IL-17, TGF-β)
Cytokine concentration

dependent variables

Id1 protein levels in the collected media (analyzed by ELISA)

control variables

Cell plating density (200,000 cells/well)
Serum starvation duration (overnight)
Media collection duration (24 h)

controls

Positive control: synovial fibroblasts from RA, OA, and other diseases analyzed by ELISA
Negative control: not explicitly mentioned

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