Cd25 clone pc61

Splenocytes and axillary and brachial lymph nodes were harvested from mice and prepared into single cell suspensions. CD4⁺ T cells were purified with antibody-conjugated magnetic beads (MagniSort mouse CD4 selection kit, eBioscience; San Diego, CA) and labeled with the following fluorophore-conjugated mAbs against: CD4 (clone RM4-5), CD25 (clone PC61.5), CD62L (clone MEL-14), and CD44 (clone IM7) (eBioscience). Naïive CD4 T cells (CD4⁺ CD25⁻ CD62L^high CD44^low) were sorted on an Influx flow cytometer (BD Biosciences; San Jose, CA) or MoFlo XDP (Beckman Coulter; Indianapolis, IN) with a sort purity of greater than 98%. Alternatively, for some experiments, naive CD4 T cells were directly isolated from lymphocyte suspension via negative selection using magnetic beads (StemCell Technology; Vancouver, BC) with a purity of at least 90-95%.

Cell suspensions were stained in ice cold PBS with or without 2 % FBS on ice. The following antibodies were used for staining mouse cells: CD45 (clone 30-F11, Biolegend), Ly6G (clone 1A8, BD Biosciences), CD11c (clone HL3, BD Biosciences), CD11b (clone M1/70, Biolegend), Siglec F (clone E50–2440, BD Biosciences), CD3e (clone 145–2C11, eBioscience), CD4 (clone GK1.5, Biolegend), IFN © (clone XMG1.2, Biolegend), IL-17 (clone eBio17B7, eBioscience), IL-5 (clone TRFK5, Biolegend), IL-13 (clone eBio13A, eBioscience), CD25 (clone PC61.5, eBioscience), CD44, CD62L (clone MEL-14, BD Biosciences), MHC class II I-A/I-E (clone M5/114.15.2, eBioscience), DC-SIGN/CD209 (R&D systems), CD32 (clone D34–485, eBioscience), TLR4/CD284 (clone SA15–21, Biolegend), Annexin V (Biolegend). Neutrophils were defined as CD45⁺CD11b⁺Ly6G⁺DNA⁺ and cytoplasts were defined at CD45⁺CD11b⁺Ly6G⁺DNA⁻. Naïve T lymphocytes were defined as CD4⁺CD25⁻CD44^lowCD62L^hi. Lung Dendritic cells were defined as CD45⁺CD11c⁺ MHCII⁺autofluorescence^low. The following antibodies were used to stain human cells: anti-CD45 PE-Cy7 (HI30), anti-CD66b (G10F5), anti-CD16 APC-Cy7 (3G8) all from Biolegend. Vybrant DyeCycle Ruby (Life Technologies) was used to stain intracellular DNA. Data were acquired on BD Canto II or BD LSR Fortessa and analyzed using FlowJo v10. For cell sorting, FACS Aria was used.