Anti cd11c clone n418

To assess FcγR dependent BMDC maturation, ova ICs were added directly to culture wells on day 6–7 of culture; ova or rabbit anti-ova alone were used as controls. 24 hours later, BMDCs were harvested, washed, blocked with a non-fluorescent FcγRII/III antibody (clone 2.4G2, BioLegend) and stained for cell surface expression of CD54, CD80, CD86 and MHCII I-A^b (clones YN1/1.7.4, 16-10A1, GL-1 and AF6-120.1 respectively, BioLegend). To identify live DCs, cells were also stained with anti-CD11c (clone N418, BioLegend) and a viability marker (Zombie Fixable Viability dye, BioLegend). Cells were washed with FACS buffer (5% FBS, 1 mM EDTA in PBS) and fixed using 1% paraformaldehyde (PFA, Electron Microscopy Sciences) in PBS prior to flow cytometry. Maturation marker expression was determined by gating on live CD11c⁺ singlets.
To determine FcγR induced cytokine production, BMDCs were harvested on day 7 of culture, counted and replated in 96 well round bottom plates at 2 × 10⁵/well, and stimulated with ova, rabbit anti-ova, ova pre-incubated with rabbit IgG, or ova ICs for 3–24 hours, in the presence of polymyxin B (50 µg/ml, Sigma). Cell-free supernatants were removed and assessed for IL-6, TNFα, and IL-12/23p40 cytokine expression by immunoassay as per the manufacturer’s instructions (BioLegend).

Tumors enzymatically digested by ± 2.3 Wunsch units / ml Liberase TL (Roche, Basel, Switzerland) and tumor draining lymph nodes were passed through 70 μm cell strainers. The generated single cell suspensions were stained with the live/dead marker Zombie Aqua (Biolegend, San Diego, CA, USA) and subsequently blocked for unspecific binding to CD16/32 (TruStain fcX, Biolegend). In order to investigate different myeloid cell populations and endothelial cell activation the following antibodies were diluted in FACS buffer (1% FCS, 0.02% NaN₃ and 3 mM EDTA in PBS): anti-CD11b (clone M1/70, Biolegend), anti-CD11c (clone N418, Biolegend), anti-B220 (clone RA3-6B2, Biolegend), anti-CD86 (clone GL-1, Biolegend), anti-CD45 (clone 30-F11, Biolegend), anti-Gr1 (clone RB6-8CS, Biolegend), anti-Ly6C (clone HK1.4, Biolegend), Ly6G (clone 1A8, Biolegend), ICAM-1 (clone YN1/1.7.4, Biolegend), VCAM-1 (clone MVCAM.A, Biolegend) and CD31 (clone MEC 13.3, BD Biosciences, Franklin Lakes, NJ, USA). Samples were washed with FACS-buffer and analyzed in a FACSCanto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was performed with FlowJo software (TreeStar, Ashland, OR, USA).

For phenotypic analysis cell from BMDC cultures were harvested by repeated pipetting and centrifuged at 350 g to include moderately adherent and smaller cells. The cells were then blocked one ice in FACS buffer (1% FBS and 0.5 mM EDTA in PBS) with anti-CD16/CD32 antibody (Fc Block, BD Pharmingen). Cells were then stained with anti-CD11c (clone N418, Biolegend), CD11b (clone M1/70, Biolegend), MHCII I-A/I-E (clone M5/114, Invitrogen), CD40 (clone 1C10, Invitrogen), CD80 (clone 16-10A1, Invitrogen) and/or CD86 (clone GL1, Invitrogen) antibodies in FACS buffer, fixed with 2% PFA and analyzed on a BD LSRFortessa flow cytometer (BD Biosciences). For maturation experiments BMDCs were challenged with freshly egressed T. gondii tachyzoites (PRU-RFP, MOI 1, infection frequencies ranged between 15 and 30%) or 100 ng/mL LPS or left unchallenged for 24 h.