Softmax pro software version 5

Rheumatoid factor, anti-cyclic citrullinated peptides, CRP and ESR were analysed by routine in-house methodology. The samples were treated as uniformly as possible, centrifuged within 1 h after sampling, and plasma/serum was removed and put in the freezer as soon as possible and carefully thawed in a refrigerator overnight before the analysis. All samples from the same patient (and respective control subject) were thawed at the same time for the analyses of the biomarkers. Laboratory personnel (KAB, MS and HHN) were blinded to the results of the clinical, US and laboratory assessments. Calprotectin was analysed in EDTA-plasma by a commercial enzyme-linked immunosorbent assay (ELISA) (CALPRO AS, Lysaker, Norway), and S100A12 was analysed in serum by ELISA (CycLex Co., Nagano, Japan), both according to the instructions of the manufacturers. The plates were read by an Emax plate reader with Softmax Pro software version 5.4 (Molecular Devices, Sunnyvale, CA, USA). IL-6 and VEGF were analysed in serum by a premixed multi-analyte kit (R&D Systems, Minneapolis, MN, USA), following the instructions of the manufacturer, and the plates were read on a Luminex 100 system (Luminex Corp., Austin, TX, USA) with use of STarStation version 3.0 software (Applied Cytometry Systems, Dinnington, UK).

BMDMs were lysed with 1% Triton X-100 in PBS and OmniMMP Fluorogenic Substrate (400 µM, BML-P126-0001, Enzo Life Sciences, Farmingdale, NY, USA) was added in the 1× Omni-buffer (50 mM HEPES, 10 mM CaCl₂ in dH₂O, pH 7.0). Fluorescence was measured at 2 min intervals from 0–300 min at an excitation of 320 nm and emission of 405 nm with a SpectraMax M2 and SoftMax Pro Software Version 5 (Molecular Devices).