Anti hamster igg

For flow cytometry cell sorting, cells were stained with anti-CD4 (RPA-T4) and anti-CD3 (SK7) antibodies (BioLegend); anti-CD4 (RPA-T4), anti-CD3 (SK7) and anti-HLA-DR (L243) or with anti-CD4 (SK3), anti-CD3 (UCHT1), and anti-CD14 (63D3) according to the sorting strategy. Gating strategies are depicted in Supplementary Figs 1–3. Sorted populations cell purity was routinely >98%. For intracellular cytokines assays sorted CD3^highCD4^high, rested for at least 3 h, were stimulated with 5 μg mL⁻¹ of anti-CD3 (UCHT1, BioLegend) and 2 μg mL⁻¹ of anti-ICOS (C398.4 A, BioLegend), crosslinked with 5 μg/mL anti-mouse IgG1 (BioLegend) plus 10 μg mL⁻¹ anti-hamster IgG (Thermo Fisher Scientific) at 37°C in the presence of Brefeldin-A (Life Technologies) for 14 h. Cells were fixed in paraformaldehyde 1% (Sigma-Aldrich) and permeabilized with saponin (Carl Roth). Antibodies used are listed in Supplementary Data 2. When indicated 1.7 μg mL⁻¹ LPS (Sigma-Aldrich), TNC (Merck Millipore), or cell-depleted synovial fluid (SF) was added. For TLR4 blocking, CLI-095 (InvivoGen) was added at 10 μg mL⁻¹ 1 h before stimulation. Cell sorting was performed in a BD FACSAria III instrument (BD Biosciences).