Il 13 bv421

In vitro differentiated and purified blood memory T_H17 cells were restimulated during 3 h with PMA and ionomycin using the Cell Stimulation Cocktail (eBioscience) and then stained with Alexa647-IL26, Alexa488-IL17A, Alexa568-IL22 and Alexa750-IFNγ probes using PrimeFlow RNA Assay (eBioscience) according to the manufacturer’s protocol. In some experiments, intracellular cytokine staining of re-stimulated T_H17 cells and blocked for secretion by Brefledin A after 1 h was performed using anti-human IL-9 PeCy7 (BioLegend, 1/20), IL-10 PE (BD Biosciences, 1/20), IL-13 BV421 (BioLegend, 1/20) and IL-21 PE (BioLegend, 1/20) antibodies before proceeding with the hybridization step of the Prime-Flow Assay. Cells were acquired on a FACS LSR II SORP (BD Biosciences) and analyzed with FlowJo_v10.7.1. A detailed gating strategy for this analysis is shown in Supplementary Fig. 8.

Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (Beckman Coulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-FITC/PE, CD4-FITC/APC, IFN-γ-PE-CY7/PE, TNF-α-PE-CY7, IL-4-PE/PE-CY7/BV421, IL-5-BV421, IL-13-BV421, T-bet-PE-CY7 and GATA-3-BV421 (Biolegend, UK). Anti-human antibodies NR2F2(Abcam, UK) were binding with fluorescent agent APC (Biolegend, UK) according to the manufacturer’s introduction. For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (Biolegend, UK). To detect intracellular cytokines, CD4⁺T cells were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).