Apc cy7 conjugated anti il 17a

Alexa488-conjugated anti-GATA3, Alexa647-conjugated anti-CXCR5, APC-Cy7-conjugated anti-CD4, BUV395-conjugated anti-IFNγ, BV711-conjugated anti-IL-2, Pe-Cy7-conjugated anti-CD25, PE-conjugated anti-CCR6 and anti-mouse IgG1, and PerCpCy5.5-conjugated anti-CD127 and anti-Tbet were purchased from Becton Dickinson. Alexa488-conjugated anti-IL-10, eFluor660-conjugated anti-IL-21, FITC-conjugated anti-CD45RA, PE-conjugated IL-22, Pe-Cy7-conjugated anti-IL-4 and mouse IgG1 were purchased from eBiosciences. APC-Cy7-conjugated anti-IL-17A, BV421-conjugated anti-CXCR3, and BV605-conjugated anti-TNFα was purchased from Biolegend. FITC-conjugated anti-CCR7 and recombinant human IL-12 was purchased from R&D Systems. Anti-DOCK8 mAb was purchased from Santa Cruz Biotechnology. Recombinant human TGFβ, IL-1β, IL-6, IL-21 and IL-23 were from Peprotech. Prostaglandin E2, PMA, calcium ionophore (ionomycin), Brefeldin A, and saponin were purchased from Sigma-Aldrich and recombinant human IL-4 was provided by Dr Rene de Waal Malefyt (DNAX Research Institute, Palo Alto, CA). T cell activation and expansion (TAE) beads (anti-CD2/CD3/CD28) were purchased from Miltenyi Biotec and CFSE was purchased from Invitrogen.

CD4⁺T-cells were purified (>90% purity) from whole blood using RosetteSep CD4⁺enrichment antibody cocktail (StemCell Technologies) according to manufacturer’s instructions. Cells were labeled with Pacific Blue-conjugated anti-CD4, fluorescein isothiocyanate (FITC)-conjugated anti-CD45RA, phycoerythrin (PE)-Cy7-conjugated anti-CCR4, PE-conjugated anti-CTLA4, allophycocyanin (APC)-Cy7-conjugated anti-IL-17A, PE-Cy7-conjugated anti-CD25, PE-conjugated anti-Ki67, APC-conjugated anti-CD161 (BioLegend); Alexa Fluor 488-conjugated anti-Foxp3 and Alexa Fluor 700-conjugated anti-Foxp3. Foxp3Δ2 was detected using clone PCH101 (eBiocience) that recognizes the N-terminus portion of the protein and clone 150D (BioLegend) that recognizes the exon 2 [24 (link), 25 (link)]. Data were acquired on a LSRFortessa cell analyzer (BD) and analyzed with FlowJo software.