Anti cd11c n418

Cells were acquired on a FACSCanto II (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo software (TreeStar). Cell sorting was performed on a MoFlo Astrios (Beckman Coulter, Krefeld, Germany). The following antibodies were used for staining cells: anti-CD45 (clone 30-F11, eBioscience, San Diego, USA), anti-CD11b (clone M1/70, eBioscience, San Diego, USA), anti-Gr1 (clone RB6-8C5, Biolegend, Fell, Germany), anti-Mac3 (clone M3/84 Biolegend, Fell, Germany), and anti-CD11c (N418, eBioscience San Diego, USA). Before surface staining, dead cells were stained using the Fixable Viability Dye eFluor^® 506 (eBioscience, San Diego, USA) followed by incubation with Fc receptor blocking antibody CD16/CD32 (clone 2.4G2, BD Bioscience, Heidelberg, Germany).

293T cells were transfected using Effectene (Qiagen) with the indicated plasmids and grown in complete DMEM composed of DMEM (Life Technologies) supplemented with 10% FCS, 1 x sodium pyruvate, 1 x GlutaMAX, and 1 x penicillin-streptomycin solution (Sigma). All other cells were cultured in complete RPMI composed of RPMI (Life Technologies) supplemented as complete DMEM with the addition of 52μM 2-ME. Total T cells were purified from the spleens of indicated mice after red cell lysis using the EasySep Mouse T cell isolation Kit (Stemcell Technologies). For activation assays, cells were either cultured with 10ng/ml IL-7 (Peprotech) or 2μg/ml anti-CD3 (145-2c11, eBioscience) and 10μg/ml anti-CD28 (37-51, eBioscience) for up to 48hrs. BMDCs were generated by culturing the bone marrow from indicated mice at 2x10⁵ cells/ml in complete RPMI with 20ng/ml GM-CSF (Peprotech). After 4 days the media was topped up with half the initial volume of the same media and the cells in suspension harvested after 7-8 days. Expression of CD11c was confirmed by flow cytometry (anti-CD11c, N418, eBioscience). For activation assays, 0.5x10⁶ cells were cultured in 1 ml in a 48-well plate with either 20ng/ml GM-CSF alone or with the addition of 100ng LPS (Enzo) for 48hrs.