Quicklysis solution

Peripheral blood of NOD-SCID gamma mice was drawn from the subclavian vein after an intraperitoneal injection of a sublethal dose of anaesthesia (Avertin 0.2%, 0.16 ml/g [Sigma-Aldrich; St. Louis, MO, USA]) and collected in EDTA BD microtainer tubes (BD Biosciences; Franklin Lakes, NJ, USA). All mice were killed by cervical dislocation. After removing red blood cells using Quicklysis solution (Cytognos SL; Salamanca, Spain), neutrophils were isolated and purified following the Neutrophil Isolation Kit protocol (Miltenyi Biotec; Bergisch Gladbach, Germany [reference # 130-097-658])^{17 (link)–19} . The degree of the neutrophil purity achieved (CD11b + Ly6G+ cells; Table S3 in Supplementary Material) was assessed using standard sorting procedures by flow cytometry.

Whole blood of HDs and RA patients was cultured in RPMI 1640 medium, LPS (1 μg/ml) or LTA (10 μg/ml) in the presence of 25 μl of metalloprotease TNFα–converting enzyme (TACE) inhibitor (Cytognos, Salamanca, Spain). After 4 hours in culture, cells were stained with anti-CD14-FITC and anti-TNFα-PE (BioLegend). Samples were then incubated with 2 ml of QUICKLYSIS™ solution (Cytognos). Membrane TNFα (mTNFα) was analyzed on CD14+ cells by flow cytometry. Adalimumab injections blocked mTNFα, and only free mTNFα was detected. When patient serum (and consequently adalimumab) was washed out of Toll-like receptor (TLR) ligand cultures, mTNFα was detectable (data not shown).