Anti il 17 pe cyanine7

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).

The anti-CD4-FITC (#11-0041-81), anti-IFN-γ-APC (#17-7311-81), anti-IL-4-PE (#12-7041-81), anti-IL-17-PE-Cyanine7 (#25-7177-80), anti-CD25-APC (#17-0251-81), and anti-Foxp3-PE (#12-5773-80) were produced by eBioscience, San Diego, CA, USA. The RIG-I (CST#3743S) and NF-κB p65 (CST#8242P) antibodies were produced by Cell Signaling Technology Inc., Danvers, MA, USA.