Pe conjugated anti il 17

The following mAbs and reagents were used in this study: Allophycocyanin (APC)-Alexa Fluor 750-conjugated anti-CD3, phycoerythrin (PE)-Cy5-conjugated anti-CD161, fluorescein isothiocyanate (FITC)-conjugated anti-TCR γδ, FITC-conjugated anti-IFN-γ, PE-conjugated anti-IL-17, PE-Cy7-conjugated anti-TNF-α, PE-conjugated anti-CCR6, PE-conjugated anti-CCR9, PE-conjugated anti-CXCR3, PE-conjugated anti-CXCR6, FITC-conjugated anti-perforin, FITC-conjugated anti-granzyme, FITC-conjugated anti-CD107a, FITC-conjugated mouse IgG isotype, PE-conjugated mouse IgG isotype and PE-Cy7-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA) and APC-conjugated anti-TCR Vα7.2 (BioLegend, San Diego, CA). Cells were stained with combinations of appropriate mAb for 20 minutes at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (Beckman Coulter, Brea, CA).

The following mAbs and reagents were used in this study: Allophycocyanin (APC)-Cy7-conjugated anti-CD3, phycoerythrin (PE)-Cy5-conjugated anti-CD161 and fluorescein isothiocyanate (FITC)-conjugated anti-TCR γδ, FITC-conjugated anti-CD3, FITC-conjugated anti-IFN-γ, FITC-conjugated annexin V, PE-conjugated anti-CD3, PE-conjugated anti-IL-17, PE-Cy7-conjugated anti-TNF-α, PE-conjugated anti-CD69, FITC-conjugated mouse IgG isotype, PE-conjugated mouse IgG isotype and PE-Cy7-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA); PE-conjugated anti-programmed death-1 (anti-PD-1; eBioscience, San Diego, CA) and APC-conjugated anti-TCR Vα7.2 (BioLegend, San Diego, CA). Cells were stained with combinations of appropriate mAbs for 20 minutes at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (version 1.1; Beckman Coulter, Brea, CA).

Cells were harvested, washed extensively, and dissociated into a single cell suspension. For Th17, IL-17A without CD8 expression, lymphocytes were detected as described previously. Cells were stimulated for 4 hours with 2 μg/ml leukocyte activation cocktail (BD Pharmingen™, USA) at 37°C and 5% CO₂. Upon harvest, cells were surface stained with antibody anti-human CD8a-APC (BD Pharmingen™, USA) at room temperature in the dark and then fixed and permeabilized using intracellular fixation and permeabilization (BD Pharmingen™, USA). Following fixing and permeabilization, cells were incubated with PE-conjugated anti IL-17 (BD Pharmingen™, USA). For Treg, Foxp3-producing CD25 lymphocytes were detected as described previously. Cells were surface stained with antibody CD25-APC (BD Pharmingen™, USA) at room temperature in the dark and then fixed and permeabilized using intracellular fixation and permeabilization (BD Pharmingen™, USA). Following fixing and permeabilization, cells were incubated with PE-conjugated anti-Foxp3 (BD Pharmingen™, USA). Samples were run on a flow cytometer (ACEA NovoCyte, USA). Data were analysed using FlowJo software (FlowJo, USA) and NovoExpress software (ACEA Biosciences, USA).