Multiparametric phenotyping of lymphocytes

Peripheral blood mononuclear cells (PBMCs), peripheral lymph nodes, spleen, and bone marrow cells were labeled with a combination of the following mAbs: CD3 PerCP (SP 34-2), CD4 PerCP (L-200), CD8 PerCP (SK1), CD8 APC (SK1), CD95 FITC (DX2), CD95 APC (DX2), and CD28PE (CD28.2) (BD Pharmingen, San Jose, CA). Lymphocytes from the animals treated with anti-CD8 mAbs were stained with anti-CD8-PE (DK25, Dako, Inc., Carpenteria, CA). For chimerism analyses, we used an anti-MHC class I HLA mAb (H38, One Lambda, Inc, CA) reacting specifically with donor MHC class I antigens. To assess intracellular protein expression of FOXP3 (PCH101), Ki67 (B56) and CD152 (BNI3), cells were permeabilized using fixation/permeabilization solution (eBioscience). The fluorescence of the stained samples was analyzed using a FACS Calibur flow cytometer and FlowJo software. For assessing memory cell function, fresh PBMCs were gated on lymphocytes and sorted into CD16⁻CD95⁻CD28⁺ naïve and CD16⁻CD95⁺CD28^low/high memory T cell populations using a FACS Vantage cell sorter (BD Immunocytometry System). The purity of sorted cells was consistently > 95%, as previously described (32 (link)).