Dynal cd4 t cell isolation kit

Peripheral blood mononuclear cells, CD14+ monocytes and moDCs were isolated and cultured as described ^{18 (link)}. CD4⁺ T cells were isolated using the negative selection Dynal CD4⁺ T cell isolation kit (Invitrogen). Purified CD4⁺ T cells and moDCs were used either directly in stimulation assays or co-cultured as indicated in an allogeneic mixed lymphocyte reaction (MLR) for 72 h. The various cell populations were stimulated for the indicated time-points with: purified lipopolysaccharide from Escherichia coli Serotype 0111:B4 (100 ng/ml, Sigma-Aldrich, St. Louis, MO), adenosine 5’-triphosphate disodium salt (ATP, 5 mM, InvivoGen, San Diego, CA), soluble anti-CD3 antibody (500 ng/ml; Sanquin, the Netherlands), plate-bound anti-CD3 antibody (clone OKT3 – 0.1,1, 10 µg/ml, Biolegend, San Diego, CA), anti-CD3/CD28 beads (bead-to-cell ratio 1:2 or 1:4, Invitrogen), Staphylococcal enterotoxin B (SEB, 0.05 µg/ml, Sigma-Aldrich or List Biological laboratories Inc, Campbell, CA), IL-10 (25 ng/ml, R&D Systems), IL-1β (10 ng/ml, R&D Systems), neutralizing IL-1β antibodies (canakunimab, 10 µg/ml), IL-1 receptor antagonist (anakinra, 5 µg/ml), neutralizing human IL-12p70 antibody (10 µg/ml, R&D systems, Minneapolis, MN) or appropriate isotype controls.