Jes3 12g8

Monocytes were purified from human peripheral blood mononuclear cells by positive CD14 selection (Miltenyi Biotec, Auburn, CA) or by adhesion, tested for purity and cultured with 10 ng ml⁻¹ macrophage colony-stimulating factor (M-CSF; Shenandoah Biotechnology, Warwick, PA; for MDM differentiation) or 40 ng ml⁻¹ GM-CSF and 40 ng ml⁻¹ IL-4 (R&D Systems Inc., Minneapolis, MN; for MDDC differentiation) as in ref. 42 (link). Cultured myeloid cells were treated with MDP (Bachem, King of Prussia, PA), Pam3Cys (EMD Millipore, Billerica, MA), lipid A (Peptides International, Louisville, KY), TriDAP or flagellin (Invivogen, San Diego, CA). In some experiments, MDMs were treated with polyphenol oxidase inhibitors (5 μM kojic acid, 1 mM salicylhydroxamic acid (VWR, Radnor, PA)) or a Syk inhibitor (1 μM; ThermoFisher Scientific, Waltham, MA). Supernatants were assayed for TNF (clones MAb1 and MAb11, diluted 1:1,000), IL-6 (clones MQ2-13A5 and MQ2-39C3, diluted 1:1,000), IL-8 (clones G265-5 and G265-8, diluted 1:1,000), IL-10 (clones JES3-9D7 and JES3-12G8, diluted 1:1,000; BD Biosciences) or IL-1β (clones CRM56 and CRM57 diluted 1:1,000; eBioscience, San Diego, CA) by ELISA. Myeloid cells (CD11c purity >75%) were isolated as in ref. 51 (link) from colonic resection specimens from uninvolved intestine in seven non-IBD patients undergoing surgery for diverticular disease or colon cancer.

Cytokine levels in supernatants were measured by ELISA, using antibody pairs for the following cytokines: IL-1β (CT213-c; 1:400 and CD213-d; 1:400), IL-6 (CD205-c; 1:200 and CD205-d; 1:200), IL-13 (QS-13; 1:200 and LM-1; 1:100), IL-23 (CD517-c; 1:400 and C8.6; 1:4,000), IFN-γ (MD2; 1:500 and MD1; 1:500; all from U-CyTech), IL-10 (JES3-9D7; 1:1,000 and JES3-12G8; 1:1,000; BD Pharmingen), TNF-α (MAb1; 1:500 and MAb11; 1:250) and IL-17A (eBio64cap17; 1:1,000 and eBio64dec17; 1:1,000; both from eBioscience).

Cytokine levels in supernatant were measured by ELISA, using antibody pairs for the following cytokines: IL-1β (CT213-c; CD2013-d), IL-6 (CD205-c; CD205-d), IL-13 (QS-13; LM-1), IL-23 (CD517-c; C8.6) IFN-γ (MD2; MD1; all U-CyTech), IL-10 (JES3-9D7; JES3-12G8; BD Pharmingen), TNF (MAb1; MAb11), and IL-17A (eBio64cap17; eBio4dec17; both eBioscience). IL-22 was measured using ELISA kit (88-7522-88, Thermo Fisher Scientific). Please note that absolute cytokine levels showed great donor-to-donor variation.