Cd11c n418

Tetramer-enriched samples were stained for surface markers for 30 minutes on ice using antibodies to: CD4 (GK1.5, BD), CD8 (53–6.7, BD), CD90.1 (HIS51), CD90.2 (53–2.1, 30-H12), CD3e (145-2C11, BD), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7, BD), CD45.1 (A20, Biolegend), CD45.2 (104), B220 (RA3–6B2), and/or PD1 (J43). Cellular viability was confirmed using GhostDye Red 780 (Tonbo Biosciences). For transcription factor expression analysis, stained cells were fixed and permeablized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Cells were stained overnight at 4°C with antibodies to T-bet (4B10, Biolegend), Foxp3 (FJK-16s), RORγt (Q31-378, BD), and Bcl-6 (K112-91, BD). For thymic dendritic cell and thymic epithelial cell analyses cells, following enrichment cells were stained with antibodies to CD11c (N418), CD19 (6D5, Biolegend), I-A/I-E (M5/114.15.2, Biolegend), CD8 (53–6.7, BD), CD90.2 (53–2.1, 30-H12), CD11b (M1/70), and CD45.2 (104, BD). Antibodies were purchased from eBioscience unless otherwise indicated. Cells were analyzed by flow cytometry on a Fortessa (BD). Data were analyzed using FlowJo software (TreeStar).

Peripheral blood mononuclear cells (PBMCs) were collected in 20 mM EDTA in Eppendorf tubes. PBMCs were spun for 8 minutes at 10,000 rpm at 20°C and serum was removed. RBCs were lysed with ACK Lysis buffer (GIBCO) for 8 minutes at room temperature (rt). If incomplete lysis occurred, cells were spun down for a second time at 12,000 rpm for 30 s and the ACK lysis step was repeated. Mononuclear cells were stained 1:100 with CB_101–109/H-2D^b-PE tetramer in the presence of Fc block 1:100 (αCD16/32, Tonbo), and monoclonal antibodies at 1:100 specific to CD45 (30-F11, Biolegend), CD8α (53–6.7, ebioscience), LFA-1 (clone H155–78, Biolegend), CD44 (clone IM7, BD), CD62L (MEL-14, Biolegend), PD-1 (J43, Invitrogen), KLRG1 (2F1, Biolegend), Tim-3 (RMT3–23, Biolegend), Lag-3 (C9B7W, Biolegend). CD19 (1D3, BD), F4/80 (T45–2342, BD), and CD11c (N418, BD) were used for a dump channel along with ghost dye BV510 at 1:500 (Tonbo). Antibodies were diluted in FACs buffer (PBS + 2.5% FBS) and cells were stained at room temperature in the dark for 60 minutes and then added to 100 μl of cell counting beads (Sigma Aldrich). Stained cells were fixed with 0.4% paraformaldehyde and stored overnight in the dark at 4°C prior to FACs acquisition using a Fortessa 1770 flow cytometer and Facs Diva software (BD Biosciences) and analyzed using FlowJo software (version 10).

Spleen sections (10 μm in thickness) were fixed in ice-cold acetone and blocked with Fc-Block (CD16/CD32) in PBS 0.1% BSA prior to incubation with Alexa Fluor 488-conjugated antibody to mouse GL7 (GL7, Biolegend), APC-conjugated IgD (11-26c.2a, eBioscience) and counterstained with DAPI (Sigma D8417). For detection of immune complex deposition in the kidneys, kidneys were mounted in OCT and 6 µm thick frozen sections were blocked with normal horse serum (Vector S-2000) then stained against IgM and IgG2c whilst nuclei were DAPI stained
Aortic arches were harvested, removed surrounding fat and adventitia and fixed in 4% PFA. After they were cut open longitudinally the arch containing lesser and greater curvature was pinned on a parafilm bed. Spleen sections (10 µm in thickness) were fixed in 4% PFA. Permeabilisation was performed with 0.2% Triton X-100 in PBS. Blocking was carried out with normal horse serum followed by primary antibody (CD11c N418, biotin, Pharmingen), and secondary antibody (Streptavidin Dylight 488 conjugated (Vector SA-5488)) staining. Subsequently tissue was stained with Nile red (Sigma N3013)and finally DAPI. Confocal images were taken using a Leica TCS inverted microscope.