Pe cy7 labeled anti cd4

Monoclonal antibodies used for phenotypic and intracellular cytokine analyses: isothiocyanate (FITC)-anti-CD3, PE-Cy7-labeled anti-CD4, and peridinin-chlorophyll protein (PerCP)-labeled anti-CD8 were purchased from BD Biosciences (San Jose, CA). Rat ELISA kits for cytokine IFN-γ and IL-4 were purchased from BD Biosciences.

Cells were washed and suspended in 100 μL of PBS containing 0.1% BSA and 0.05% sodium azide. For surface staining, cells were incubated with the respective mAbs at 4°C in the dark for 30 min. For the detection of intracellular cytokines, cells were fixed with 4% paraformaldehyde and permeabilized in PBS buffer containing 0.1% saponin (Sigma-Aldrich, USA), 0.1%BSA and 0.05% sodium azide for at least 2 h or overnight at 4°C and stained with conjugated mAbs for intracellular cytokines. Flow cytometry data were acquired with FACS Canto II (BD Bioscience, USA) and analyzed with FlowJo software (Tree Star, USA). The following mAbs were used for cell surface or intracellular staining: FITC labeled anti-CD3, anti-CD45RA, anti-Perforin, APC-labeled anti-CD69, anti-TNF-a, anti-PD-1, PE-labeled anti-CD103, anti- IFN-γ, anti-GranzymeB, Percp labeled anti-CD3, PE-cy7 labeled anti-CD4, Apc-cy7 labeled anti-CD8 and isotype matched control antibodies were all purchased from BD Bioscience PharMingen (San Jose, CA, USA).