Anti cd4 rm4 5

After homogenizing spleens, cells were treated with erythrocyte lysis buffer, meshed through a 35-μm cell strainer, washed with PBS, and analyzed by FACS.
DCs were phenotyped by using fluorochrome-labeled mAbs directed against CD11c (N418; BioLegend), CD80 (16-10A1; BD Pharmingen, Heidelberg, Germany), CD86 (PO3; Thermo Fisher Scientific), PD-1 (J43, Thermo Fisher Scientific), CTLA-4 (UC10-4B9; BioLegend), CD83 (Michel-19; BioLegend), CD40 (3/23; BioLegend), MHCII (2G9; BD Pharmingen) and PD-L1 (MIH6; AbD Serotec, Puchheim, Germany). Dead cells were excluded using the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Following mAb labeling for 30 min at 4 °C and final washing, cells were analyzed on an LSR II flow cytometer.
To measure intracellular cytokines, cells were stimulated with PMA and ionomycin (1 μg/ml each; Sigma-Aldrich, Taufkirchen, Germany) for 4 h in the presence of 3 μg/ml Brefeldin A (Thermo Fisher Scientific), labeled for CD11c, subjected to fixation and permeabilization and subsequently stained with mAbs against IL-10 (JES5-16E3; BD Pharmingen) and IL-12 (C15.6; BD Pharmingen). IFN-γ in T cells was measured by using fluorochrome-labeled anti-IFN-γ (XMG-1.2, BioLegend) and counterstaining with anti-CD4 (RM4-5, eBioscience, Frankfurt, Germany).

Mouse splenocytes and thymocytes were prepared from pooled thymus or spleen. Thymic and splenic cells were pre-incubated with Fc-block before staining with other antibodies. The mouse antibodies used included anti-CD4 (RM4–5, eBioscience); anti-CD8 (53–6.7, eBioscience); anti-CD45.1 (A20, BioLegend), anti-CD45.2 (104, eBioscience); anti-CD25 (PC61.5, eBioscience); anti-CD69 (H1.2F3, eBioscience); anti-CD24 (M1/69, eBioscience); anti-Helios (22F6, eBioscience) before flow cytometry analysis. For intracellular staining of Ki-67 (B56, BD), pSTAT5 (47/Stat5(pY694), BD) and Foxp3 (NRRF-30, eBioscience), cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation / Permeabilization Solution Kit (554,714, BD) and stained according to the manufacturer’s protocols. The CD4⁺ T cells from the thymus and spleen of WT or RelB^−/− mice were analyzed by flow cytometry and gated as shown in the Supplementary Figure 1. The samples were analyzed using a BD LSRFortessa flow cytometer and FlowJo software (Tree Star Inc). All single-cell suspensions from the tissues were stained with Abs diluted in PBS containing 2% FCS for 30 min on ice.

dLN and spleen were harvested into PLP buffer (0.05 M phosphate buffer containing 0.2 mL lysine (pH 7.4), 2 mg ml⁻¹ NaIO₄, 10 mg ml⁻¹ paraformaldehyde), fixed overnight and dehydrated in 30% sucrose prior to embedding in OCT freezing media (Sakura Fineteck). Frozen sections (10 µm) were cut on a CM1950 Cryostat. Sections were stained in PBS (0.01% Triton X-100 and 5% goat serum) using the following Abs: anti-B220 (RA3-6B2, 1:200, eBioscience) and anti-CD4 (RM4-5, 1:200, eBioscience), CD45.1 (A20, 1:100, BD Biosciences). Images were acquired on a Apotome ZEISS Inv. Regions and cells were defined with IMARIS image analysis software.

Mice were euthanized by isoflorane overdose to keep their circulatory system intact. Mice were perfused with 30mls of 1X PBS. Brain was collected in 2mls RPMI+5%FBS. Brain was disassociated over a 40μm strainer using the plunger of a 3ml syringe. The strainer was washed well with 15-25mls of RPMI+5%FBS. The suspension was centrifuged at 1650 RPM, 5 mins, 4°C and the pellet resuspended in 7mls of 30% Percol in a 15ml conical tube, centrifuged at 1650 RPM, 30 mins at room temperature or 4°C. Myelin and excess percol was aspirated off and the pellet was transferred to a new 15ml conical tube and washed with 10mls of RPMI+5%FBS followed by centrifugation at 2000 RPM, 5 mins, 4°C. The pellet was then transferred in a 96 well microtitre plate for surface staining [LIVE/DEAD yellow (Life Technologies, Carlsbad, CA), anti-CD3 (2c11, eBiosciences), anti-CD4 (RM4-5, eBiosciences), anti-CD8α (53–6.7, eBiosciences), E641:I-A^b tetramer].