Phycoerythrin conjugated anti il 17a

Astrocytes and CD4+ T cells were collected by mechanical pipetting and were stimulated for 4 h with PMA (50 ng/mL; Alexis) and ionomycin (750 ng/mL; Sigma) in the presence of Brefeldin A (BFA) at the recommended concentrations (BD Pharmingen) in a 96 well plate. Standard intracellular cytokine staining was carried out as described [34] (link). Cells were first stained extracellularly with APC–conjugated anti-CD4 (eBioscience) and fixed and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) and then were stained intracellularly with eFluo450-conjugated anti-IFN-γ, phycoerythrin-conjugated anti-IL-17A, (eBioscience). Samples were acquired on a LSRII (BD) and data were analyzed with FlowJo software (Tree Star, Inc.).

To quantify IL-17A⁺, RANKL⁺, and CD25⁺ forkhead box P3 (FOXP3)⁺ cells in CD4⁺ T cells, PBMCs cultured under Th17 polarisation conditions with various doses of IL-18BP were immunostained using a PerCP-conjugated anti-CD4 antibody (BD Biosciences, San Jose, CA, USA), then fixed and permeabilised using a Cytofix/Cytoperm Plus kit (BD Biosciences). Following the manufacturer’s instructions, PBMCs were stained with phycoerythrin-conjugated anti-IL-17A (eBiosciences, San Diego, CA, USA), or phycoerythrin-conjugated anti-RANKL (eBiosciences), or allophycocyanin-conjugated anti-CD25 (BD Biosciences) with phycoerythrin-conjugated anti-FOXP3 (BioLegend, San Diego, CA, USA) antibodies. All cells were detected using a FACSCalibur flow cytometer (BD Pharmingen, Franklin Lakes, NJ, USA).