Fc receptor blocker

Peripheral blood mononuclear cells (PBMCs) were isolated from the spleen by lymphocyte separation medium according to manufacturer (MultiSciences Biotech) instructions. Different subsets of T cells were evaluated by flow cytometry. All anti-mouse-specific antibodies used were obtained from eBioscience (San Diego, CA, USA). PBMCs were stimulated with phorbol myristate acetate (12.5 μg/mL; MultiSciences Biotech) and ionomycin (0.25 mg/mL; MultiSciences Biotech) in the presence of an FC receptor blocker (MultiSciences Biotech) for 5 h. PBMCs were washed and then fixed/permeabilized in fixation/permeabilization buffers (eBioscience) and stained with antibodies against cluster of differentiation (CD)4-Phycoerythrin (PE), CD25-Allophycocyanin (APC), interleukin (IL)-4-APC, interferon (IFN) gamma-biotin, IL-17A-Alexa Fluor 488, and forkhead box P3 (Foxp3)-PE-cyanine5.5. Appropriate isotype controls were used. Flow cytometry was undertaken on a FACSVerse flow cytometer (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo v10 (FlowJo, Ashland, OR, USA).

PBMCs were isolated from the spleen by lymphocyte separation medium (TBDscience Co., Tianjin, China) according to the manufacturer’s instructions. Different subsets of T cells were evaluated by flow cytometry. All anti-mouse-specific Abs used in this study were obtained from eBioscience (San Diego, CA, USA). PBMCs were stimulated with phorbol myristate acetate (PMA, 25 ng/mL, MultiSciences Biotech Co., Ltd., Hangzhou, China) and ionomycin (1 μg/mL, MultiSciences Biotech Co.) in the presence of FC Receptor Blocker (MultiSciences Biotech Co.) for 4 h. The cells were washed and then fixed/permeabilized in the eBioscience fixation/permeabilization and permeabilization buffers and stained with anti-CD4-FITC, anti-CD25-APC, anti-IL-4-PE, anti- IFN gamma-APC, anti-IL-17-PE-Cyanine7, and anti-Foxp3-PE. Appropriate isotype controls were performed. Flow cytometry was performed on a BD FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by using FCS Express3 software (De Novo, Kiev, Ukraine).