Cd11c apc hl3

Splenocytes were prepared as described before. Dendritic cells were enriched by a pre-isolation step using NKp46, CD3e and CD19 MACS beads negative selection (all Miltenyi Biotech, Germany). Subsequently, cells were stained for CD3e PerCP-eFluor 710 (clone 17A2, Thermo Fisher Scientific), CD19 FITC (clone 1D3, BD Biosciences), CD11c APC (HL3, BD Biosciences), CD11b PE-Cy7 (clone M1/70, Thermo Fisher Scientific) and HLA-DR BV605 (clone G46-6, BD Biosciences) and sorted using a BD FACS Aria III (BD Biosciences). Nonspecific binding through Fc gamma receptors was blocked by a mixture of anti-CD16 and anti-CD32 antibodies (Fc Block; BD Biosciences). Post sort analysis using FlowJo software (Version 10.8.1; BD Biosciences) showed an average purity of 94.8% for splenic CD11c high MHC class II+ dendritic cells.

Spleens were finely minced and passed through a 70-μM nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Spleen cells were collected in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 10% preselected fetal calf serum (FCS), 2 mM l-glutamine (both from Hyclone, Logan, UT), 100 U/mL penicillin, 100 mg/mL streptomycin (both from Life Technologies), and 5 × 10–5 M β-mercaptoethanol (Sigma-Aldrich). Single-cell suspensions were cleared of erythrocytes by hemolysis using a hypotonic buffer (pH 7.2) composed of 0.15 M ammonium chloride, 10 mM potassium bicarbonate (both from Merck, Darmstadt, Germany), and 0.1 mM ethylene-diaminetetraacetic acid (Life Technologies).
To evaluate the composition of APCs, splenocytes were stained for CD3e PerCP-eFluor 710 (clone 17A2, Thermo Fisher Scientific), CD19 FITC (clone 1D3, BD Biosciences), CD11c APC (HL3, BD Biosciences), CD11b PE-Cy7 (clone M1/70, Thermo Fisher Scientific) and HLA-DR BV605 (clone G46-6, BD Biosciences) and analyzed using a BD FACS Aria III (BD Biosciences) and FlowJo software (Version 10.8.1; BD Biosciences). Nonspecific binding through Fc gamma receptors was blocked by a mixture of anti-CD16 and anti-CD32 antibodies (Fc Block; BD Biosciences).

Splenocytes were prepared as described before. Monocytes/macrophages were enriched by a pre-isolation step using NKp46, CD3e and CD19 MACS beads negative selection (all Miltenyi Biotech, Germany). Subsequently, cells were stained for CD3e PerCP-eFluor 710 (clone 17A2, Thermo Fisher Scientific), CD19 FITC (clone 1D3, BD Biosciences), CD11c APC (HL3, BD Biosciences), CD11b PE-Cy7 (clone M1/70, Thermo Fisher Scientific) and HLA-DR BV605 (clone G46-6, BD Biosciences) and sorted using a BD FACS Aria III (BD Biosciences). Nonspecific binding through Fc gamma receptors was blocked by a mixture of anti-CD16 and anti-CD32 antibodies (Fc Block; BD Biosciences). Post sort analysis using FlowJo software (Version 10.8.1; BD Biosciences) showed an average purity of 94.4% for splenic CD11b+ monocytes/macrophages.