Anti il 17a fitc

Cultured lymphocytes were harvested and labelled CD4⁺ T cells separated by OctoMACS™. CD4⁺ T cells were incubated with 5 µM MntC in RPMI-1640 for 6 h in the presence of PMA (50 ng/mL, Sigma-Aldrich), ionomycin (1 mM, Sigma-Aldrich), and Golgistop (1 µL per 1.5 mL cell culture medium, BD Pharmingen, San Diego, CA, USA) Then the cells were washed and labelled with anti-IFN-γ-PE (eBioscience, San Diego, CA, USA), anti-IL-4-PE (eBioscience) and anti-IL-17A-FITC (eBioscience) in 1 × Permeabilization Buffer (eBioscience). Approximately 1 × 10⁵ cells were acquired using the flow cytometer CytoFLEX (A00-1-1102, BECKMAN COULTER), and the data were analysed using CytExpert software.

The pre‐treated CD4⁺ T cells were washed, and the following mouse antibodies were added for intracellular staining: anti‐CD4‐PE, anti‐IL‐17A‐FITC, anti‐CD25‐FITC and anti‐Foxp3‐APC (eBioscience, San Diego, CA, USA). The cells were fixed in the 4% paraformaldehyde and were permeabilized in the 1% Triton X‐100 at 25°C. The flow cytometry was performed on a FACS Canto™ II (BD, USA). The events were recorded and then analysed using the FlowJo7.6.1 software (Tree Star, Palo Alto, CA, USA).

Anti-CD25-PE, anti-CD4-FITC, and anti-CD4-APC antibodies were obtained from BD Biosciences—Pharmingen (San Jose, CA, USA); anti-IL-17A-FITC, anti-IFN-γ-PerCP, and anti-Foxp3-APC (FJK-16s) staining kits were from eBiosciences (San Diego, CA, USA); anti-CD80-FITC, anti-CD86-APC and anti-HLA-DR-PE antibodies were obtained from BD Biosciences—Pharmingen. Phorbol 12 myristate 13-acetate (PMA), Ionomycin calcium salt and Brefeldin A (BFA) were from Sigma-Aldrich (Milano, Italy). Purified lipopolysaccharide (LPS) was purchased from Invivogen (San Giuliano Milanese, Milano, Italy). RPMI 1640, antibiotics (penicillin and streptomycin), L-glutamine, heat-inactivated fetal bovine serum were purchased from Celbio (Pero, Italy) and used for cultures with dendritic cells.