Alexa fluor 647 anti foxp3

Flow cytometry experiments were performed to assess T cells co-cultured with MenSCs. The fluorochrome-conjugated antibodies included Alexa Fluor 647 anti-FoxP3 (BD Biosciences, USA), FITC anti-human IFN-γ, PerCP/Cy5.5 anti-CD4, APC anti-human IL-10, and PE anti-CD25 (All from Biolegend, USA) were employed. Antibodies were used at the concentrations recommended by the manufacturers. For assessment of cytokines, cells were treated with 50 µg/mL PMA (Sigma, USA), 1 µg/mL ionomycin (Sigma, USA) and 0.7 µg/mL Monensin (BD Biosciences, USA) 6 h. before staining procedure. For intracellular staining of Foxp3 and cytokines, transcription factor buffer set (BD Biosciences, USA) was used for cell permeabilization and fixation according to the manufacturer’s instruction. Cells were also stained with Live/Dead fixable near red fluorescent dye (Molecular Probes, USA) to separate the alive and dead populations before antibody-specific gating. The stained cell was then read by flow cytometer Attune NxT flow cytometer and analyzed by FlowJo software (FlowJo, LLC, USA).