Flow cytometry
All anti-bodies listed were obtained from eBioscience, San Diego, CA, USA, 5ul labeled antibody was added in ench procedure. The expression of CD28, CTLA-4, CD80 and CD86 in CD4+ T Lymphocytes was assayed in PBMCs and splenocytes. Add anti-human (mouse)CD4-FITC monoclonal antibodies, anti-human(mouse) CD28-PE, anti-human(mouse) CTLA-4-APC or anti-human(mouse) CD80-PE, anti-human(mouse) CD86-APC at room temperature away from light for 30 minutes. A parallel control group was treated with isotype controls according to the manufacturer’s recommendations.
Stimulated and cultured mononuclear cells were collected, pre-incubated for 15 minutes with unlabeled isotype control Abs (IgG1 or IgG2b). Then incubated with anti-human CD3-FITC, anti-human CD8-PE, or anti-human CD4-FITC and anti-human CD25-PE, a parallel control group was treated. splenocytes incubated with anti-mouse CD4-FITC, with or without anti-mouse CD25-PE at room temperature away from light for 30 minutes, then incubated at room temperature away from light for 15 minutes after treating with 100 μL FIX & PERM medium A and B (Invitrogen, USA). Then, anti-human IL-17-APC, anti-human IFN-g-APC, anti-human IL-4-APC or anti-human FoxP3-APC was added in PBMCs and anti-mouse IL-17-APC, anti-mouse IFN-g-APC, anti-mouse IL-4-APC or anti-mouse FoxP3-APC was added in splenocytes. A parallel control group was treated d with isotype controls. Both suspensions were incubated at 4 temperature away from light for one night.