Anti cd4 apc cy7 gk1

Cell suspensions were generated from spleens and PALNs by gentle homogenization in RPMI 1640 (Gibco^TM, Life Technologies, Invitrogen, Australia Pty. Ltd.). Erythrocytes were lysed in splenocyte suspensions using red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 0.1 mM EDTA). Dead cell exclusion was enabled by addition of BD Horizon™ Fixable Viability Stain 620 (Becton Dickinson (BD), Franklin Lakes, NJ). 10⁶ cells were stained in PBS (0.05% sodium azide, 0.1% BSA (Sigma). Fc receptors were blocked with anti-CD16/CD32 Fc Block™ (Mouse Fc Block™; BD) before surface staining with antibodies; anti-CD4 APC-Cy7 (GK1.5, BD), anti-CD8α PE-Cy7 (53–6.7, BD) and Nrp1 BV421 (3E12, Biolegend, San Diego, CA). Intracellular staining was performed using Foxp3 Staining Buffer Set (eBioscience, San Diego, CA), as per the manufacturer’s instructions, and anti-Foxp3 APC (FJK-16s, eBioscience) antibody. Data were acquired on a BD FACSCantoII. All data were analysed using FlowJo software (TreeStar, Inc., Ashland, OR).

Blood samples and splenocytes from anti-CD25- or IL2-treated mice and relevant controls were stained with anti-CD3 FITC (145-2C11, BD Biosciences), anti-CD4 APC-Cy7 (GK 1.5, BD Biosciences), anti-CD8a PerCP-Cy5.5 (53–6.7, BD Biosciences), anti-CD25 APC (PC61, BD Biosciences), anti-CD44 BV421 (IM7, BD Biosciences) and anti-CD62L BV510 (MEL-14, BD Biosciences) antibodies. Cells were then fixed and permeabilized using the Foxp3/Transcription factor fixation/permeabilization Kit (Invitrogen). Cells were then additionally stained with anti-Foxp3 Alexa700 (FJK-16 s, eBioscience), anti-T-bet PE (4B10, BD Biosciences), anti-RORγT PE-CF594 (Q31-378, BD Biosciences) and anti-GATA3 PE-Cy7 (L50-823, BD Biosciences) antibodies. Alternative staining consisted in anti-CD3 FITC (145-2C11, BD Biosciences), anti-TCRγδ PE-CF594 (GL3, BD Biosciences), anti-CD19 BV421 (1D3, BD Biosciences), anti-NK1.1 Alexa700 (PK136, BD Biosciences), anti-NKp46 PE (REA815, Miltenyi), anti-CD11b PE-Cy7 (M1/70, BD Biosciences), anti-CD11c APC (HL3, BD Biosciences) and anti-Ly6G APC-Cy7 (1A8, BD Biosciences) antibodies. Cells were then fixed using CellFix reagent (BD Biosciences). All samples were processed with a Gallios flow cytometer (Beckman Coulter) and the data analyzed using the Kaluza software (Beckman Coulter).