Anti human mabs

A total of 1 × 10⁶ PBMCs were stained with the following anti-human mAbs (BD Pharmingen, San Diego, CA): CD3-Pacific Blue (558117), CD56-APC (555518) and CD127-PE (557938) for surface staining. Nonspecific binding was prevented by a short incubation with fetal bovine serum before the addition of specific antibodies. For intracellular cytokine staining, PBMCs were incubated at 37°C for 72h in a 24-well plate (1 X 10⁶ cells/well) with or without IL-7 (50 ng/ml) and followed by a mixture of PMA (20 ng/ml), Ionomycine (1 μg/ml) and GolgiPlug (1μg/ml) for another 5h at 37°C. After harvesting, cells were stained for surface markers CD3-Pacific Blue (558117) and CD56-APC (555518) followed by fixation and permeabilization with the Cytofix/Cytoperm kit (BD). They were then respectively stained with anti-IFN-γ-PE (BD, 340452) or anti-Bcl-2-PE (BD, 340651) as designed. After staining, all samples were acquired on a FACSAria^™ flow cytometer and analyzed with FACSDiva software 6.1.3 or FCS Express 4 software (BD, Mountain View, CA).