Cxcl10

Supernatants from GM-MØ were tested for the presence of IL-6, IL-10, TNFα, and CXCL10 (BioLegend) and IFNβ (R&D Systems). For the detection of sCD14 in supernatants, serum, and plasma of RA patients and healthy controls, the Human CD14 Quantikine ELISA Kit (R&D Systems) was used.

Cell culture supernatants were collected from tenocytes stimulated with 1 µg/ml S100A8 & S100A9, 10 ng/ml IL-1ß for 24 hours and the concentrations of IL-6, IL-8, CCL2 (Thermo Fisher Scientific, range 2000-31.25 pg/ml), CCL20, CXCL10 (Biolegend, CCL20 range 160-5 pg/ml, CXCL10 1000-15.6 pg/ml)), S100A8 and S100A9 (R & D systems, range 2000-31.25 pg/ml) were determined using commercially available ELISA kits. Cell culture supernatants were diluted with assay diluent to achieve concentrations within the specified range. Optical density was measured at 450 nm by a microplate reader.

CXCL10 and CXCL8 secretion was measured in the supernatant with the use of a commercially available ELISA kit (CXCL10: BioLegend, San Diego, CA, USA or R&D Systems, Minneapolis, MN, USA; CXCL8: Invitrogen, Thermo Fisher Scientific) both according to the manufacturer’s protocol. In short, high-binding 96-well plates were coated with capture antibody and incubated overnight. Non-specific binding was blocked for 1 h at room temperature. After washing, the samples or the standard were added for 2 h at room temperature. Then, plates were washed and incubated with streptavidin-horseradish peroxidase for 1 h at room temperature. Subsequently, the plates were washed and incubated in the dark with substrate solution at room temperature. The reaction was stopped with 1 M H₂SO₄ and absorbance was measured at 450 nm in a microplate reader (iMark, Bio-Rad Laboratories, Hercules, CA, USA or GloMax Discover, Promega Corporation, Madison, WI, USA).