Cd11c pe n418

Mice were euthanized by cervical dislocation. The spleen and the draining inguinal lymph node were excised and mashed onto 70-µm mesh (Corning, USA) to obtain single-cell suspensions. The peritoneal cavity was washed with 5 ml of 1 × PBS with 3% fetal calf serum (Biological Industries, Israel). Cells extracted from the peritoneal cavity were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) and subsequently stained with B220-FITC (53-7.3, eBioscience) and CD23-PE (B3B4, eBioscience) to identify B cells and with CD11c-PE (N418, eBioscience) and Gr-1-FITC (RB6-8C5, eBioscience) to identify dendritic cells (CD11c⁺Gr-1^-), macrophages (CD11c^midGr-1⁺) and neutrophils (CD11c^-Gr-1⁺). In addition, cells were stained with CD3-FITC (17A2, eBioscience) and CD49b-PE (DX5, eBioscience) to identify T cells (CD3⁺DX5⁻) and natural killer (NK) cells (CD3⁻DX5⁺). Peritoneal cells were also stained with B220-Briliant Violet 510 (RA3-6B2, BioLegend), IgLk-FITC (187.1, BD Biosciences), IgD-PE/Cy7 (11-26c, eBioscience), CD23-Briliant Violet 421 (B3B4, BioLegend), and CD5-PerCP Cy5.5 (53-7.3, eBioscience). PerCP Cy5.5 isotype control antibodies (eBR2a, eBioscience) were used to validate the specificity of the CD5 staining. Cells were analyzed using a FACSCalibur flow cytometer or a FACSAriaIII cell sorter (BD biosciences) and FlowJo software (Three Star).