Percp cy5 mouse anti human cd3

PBMCs were seeded into a 10-mL sterile tube, containing 1 mL of RPMI media (Gibco) containing penicillin/streptomycin 10.000 U/10 mg (Sigma-Aldrich), 10% fecal bovine serum (FBS) (Gibco), at a final concentration of 5 × 10⁵ cell mL⁻¹. To stain intracellular cytokines, cells were activated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), and 1.25 pM calcium ionophore (Sigma-Aldrich) during 2 h at 37 °C in a CO₂ incubator (Heraeus 6000). Then, Brefeldin A was added to the cell solution, to the final concentration of 5 μg·mL⁻¹ (Sigma-Aldrich) and incubated overnight at 37 °C, in a CO₂ incubator [7 , 21 (link)].
Cells were fixed and permeabilized with Immunostep Fix and Perm Kit, adding solution A (fixative) and incubated for 15 min at room temperature. Then, cells were washed with Facs Flow buffer (BD) and centrifugated at room temperature during 7 min at 1700 rpm in the Heraeus Labfuge 400. Solution B (permeabilization) was added to the cells, with conjugated intracellular antibody cocktail (FITC Mouse Anti Human IFN-γ (BD); PE Mouse Anti Human TNF-α (BD Pharmigen); PerCP-Cy5 Mouse Anti Human CD3 (BD); Anti IL-17A-APC Human (Miltenyi Biotec); at room temperature during 30 min in the dark). Cells were washed with Facs Flow buffer (BD) and resuspended in 1 mL Facs Flow buffer (BD). Cells were screened with Facs Calibur (BD), and analyzed with FlowJo software v8.7.

To determine PBMC transcription factor activation with flow cytometry, we used Pharmigen™ Transcription factor buffer set (BD) as recommended by the manufacturers. We seeded in a 10-mL sterile tube to a final concentration of 5 × 10⁵ cell mL⁻¹ in 1 mL of RPMI media (Gibco) and completed with penicillin/streptomycin 10.000 U/10 mg (Sigma-Aldrich) and 10% fetal bovine serum (FBS) (Gibco). Surface staining was performed with PerCP-Cy5 Mouse Anti Human CD3 (BD) during 30 min at 4 °C. Cells were fixed and permeabilized with 1 mL Fix and Perm solution at 4 °C during 40–50 min protected from light. Cells were washed twice with Perm/wash buffer, then centrifugated at room temperature during 7 min at 1700 rpm in a Heraeus Labfuge 400.
Intranuclear staining was carried out with an antibody cocktail resuspended in Perm/Wash buffer (APC Mouse Anti Human Fox-P3 (Pharmigen) PE Mouse Anti-human T-bet (BD) and Alexa Fluor 488 Mouse Anti Human Ror-γt (BD)) at 2 °C during 40–50 min in the dark. After another washing step, cells were resuspended in 1 mL Facs Flow buffer (BD). Cells were ready to be acquired by flow cytometry with Facs Calibur (BD) and analyzed with FlowJo software v8.7.