Anti cd24 m1 69

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.

Single cell suspensions (0.2–0.5 × 10⁶ cells for NIH/3T3 derived cells) were obtained by scraping cells from plates into FACS buffer [1% heat inactivated FBS in phosphate-buffered saline (PBS, 1.86 mM NaH₂PO₄.H₂O, 8.41 mM Na₂HPO₄, 150 mM NaCl)]. Cells were incubated with 0.5 μg anti-CD24 (M1/69), or Rat IgG2a κ isotype control, conjugated to APC (eBioscience, San Diego, CA, USA) for 30 min on ice, followed by three washes with FACS buffer then fixed in 4% paraformaldehyde. Data was collected with a FACS Calibur (BD Biosciences, Mississauga, ON, Canada) and analyzed using FlowJo software v10.0.5 (Ashland, OR, USA).