Anti cd16 cd32 mab clone 2.4g2

Single cell suspensions were prepared as described7 (link) and blocked with anti-CD16/CD32 mAb (clone 2.4G2; BD Biosciences, San Diego, CA) before staining with mAb or dye. PE-labeled anti-CD4 (clone GK1.5), PE- or PE-Cyanine7-labeled anti-CD11c (clone N418), PE-Cyanine7-labeled anti-PDCA-1/BST2/CD317 (clone eBio927), Alexa Fluor 700-labeled anti-CD11b (clone M1/70), PE-Cyanine5-labeled anti-CD45R/B220 (clone RA3-6B2) and PE-labeled anti-CD23 (clone B3B4) mAb and PE-labeled streptavidin were from eBioscience (San Diego, CA). PE- or FITC-labeled anti-MHC-II (I-A^d; clone AMS-32.1), Alexa Fluor 647-labeled anti-DO11.10 TCR (clone KJ1-26) and biotinylated anti-CD86 (clone GL1) mAb and Fixable Viability Stain 450 (FVS450) were purchased from BD Biosciences. Alexa Fluor 488-labeled anti-DCIR2 (dendritic cell inhibitory receptor 2; clone 33D1) and Brilliant Violet 421-labeled anti-CD8α (clone 53-6.7) mAb were from Biolegend (San Diego, CA). FITC-labeled anti-DO11.10 TCR (clone KJ1-26) mAb was obtained from Life technologies. Events were acquired on a LSR II cytometer (BD Biosciences) and data were analysed by FlowJo software (Tree Star, Ashland, OR). Cells were gated for singlets according to their forward- and side-scatter properties prior to analysis as shown in Supplementary Fig. S2a.

Single cell suspensions were prepared from spleens and from tumors by incubating the tumors cut into small pieces in 1.6 mg/ml Collagenase IV (Worthington Biochemical Corporation, Lakewood, NJ) and 0.1 % DNase (Sigma-Aldrich, St. Louis, MO) for 45 min at 37 °C, followed by meshing the tumors in a 70 μm cell strainer. CD11b⁺ cells were further purified from tumor cell suspensions by magnetic depletion of MC38-C215 tumor cells after incubation with anti-C215-biotin (10 μg/ml) and Dynabeads® Biotin Binder beads (Life Technologies) followed by positive selection of CD11b⁺ cells by MACS technology (Miltenyi).
Before incubating the cells with specific fluorochrome-labeled antibodies, Fc-receptors were blocked using anti-CD16/CD32 mAb (clone 2.4G2; BD Biosciences). The following fluorochrome-labeled antibodies were used: CD11b (clone M1/70), Ly6G (clone 1A8), Ly6C (clone AL-21), F4/80 (clone BM8), CD206 (clone C068C2), CD86 (clone GL1), MHC II (I-A/I-E; clone M5/114.15.2), CD4 (clone RM4-5), and CD8a (clone 53-6.7), purchased from BD Biosciences (San Jose, CA), eBioscience (San Diego, CA) and BioLegend (San Diego, CA). Intracellular staining of CD206 was performed using the Mouse Regulatory T cell Staining Kit from eBioscience. Flow cytometric analysis was performed according to standard settings on a FACS CantoII flow cytometer (BD Biosciences).