Anti cd3 percp5

Fresh peripheral blood was drawn from HBV-infected and healthy children, and the red blood cells were lysed using NH₄Cl lysis solution. After washing by fluorescence-activated cell sorter (FACS) buffer, leukocytes were stained with specific fluorochrome-conjugated monoclonal antibodies (mAbs) for flow cytometric analysis. For NK cell phenotypic analysis, leukocytes were stained with mAbs, including anti-CD3-PerCP5.5, anti-CD56-APC, anti-NKp30-PE, anti-NKp46-BV421, and anti-HLA-DR-FITC (BD Biosciences, USA) mAbs. For T-cell phenotypic analysis, leukocytes were stained with mAbs, including anti-CD3-PerCP5.5, anti-CD8-APC, anti-CD4-FITC, anti-CD27-PE, anti-CD45RA-BV421 (BD Pharmingen, USA), and anti-CD38-PE-Cy 7 (Biolegend, USA) mAbs. Intracellular IFN-γ-BV421 (BD Biosciences, USA) and INF-α-PE-Cy 7 (Biolegend, USA) were detected after PMA/lonomycin mixture (250×, BioLegend, USA) for 1 h and Monensin Solution (1,000×, EB eBioscience, USA) at 37°C for 4 h. Multiparameter flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter, USA), and data were analyzed using FlowJo software V10.

EDTA-treated whole blood was incubated using the corresponding monoclonal antibodies: anti-CCR6-PB, anti-PD1-PCy7, anti-CD3-PerCP5.5, anti-CXCR3-PE and anti-ICOS-APCH7 (all from BDBiosciences, New York, NY, USA); anti-CD4-KO (from Beckman Coulter, Miami, FL, USA). Proportions of Th subsets were analyzed by flow cytometry using a Navios Cytometer and Kaluza Software (Beckman Coulter, Miami, FL, USA).
According to the expression of cell surface markers in CD4+ T cells, they could be classified as: Th1 cells (CXCR3+/CCR6-), Th17 cells (CXCR3-/CCR6+) and Th2 cells (CXCR3-/CCR6-). Regarding the activation status, each one could be divided into quiescent cells (ICOS-/PD1-), early activated cells (ICOS+/PD-1-), late activated cells (ICOS+/PD-1+) and exhausted or senescent cells (ICOS-/PD-1+) [32 (link)].