Lsr aria 2 cell sorter

One-two months before kidney transplantation, PBMC were harvested from single leukapheresis products of prospective graft recipients (n=2) by Ficoll density gradient centrifugation. Anti-CD2 and -CD20 microbeads (Miltenyi Biotec) were used to positively select and remove T and B cells respectively. CD14⁺ cells (monocytes) were isolated using CD14-specific immunobeads (Miltenyi Biotec) and cryopreserved. Seven and 14 days post-transplant, FACS-stained mMDSC (CD3⁻CD20⁻HLA-DR⁻CD33⁺CD14⁺) were flow-sorted from the cryopreserved CD14⁺ cells using a LSR ARIA II cell sorter (BD Sciences).

PBMC were isolated by standard Ficoll density gradient centrifugation (GE Healthcare, Pittsburgh, PA) from freshly-drawn normal human or normal rhesus monkey heparinized peripheral blood or from cytokine-mobilized rhesus monkey leukapheresis products. Anti-CD2 and −CD20 microbeads (Miltenyi Biotec) were used to positively select and remove T and B cells respectively from the PBMC. The resulting cell population was FACS stained and the putative monocytic MDSC (CD3⁻CD20⁻HLA-DR⁻CD33⁺CD14⁺) were recovered via flow sorting using a LSR ARIA II cell sorter (BD Sciences). In addition, the flow-sorted MDSC were stained for CD11b and CD274 (B7-H1) and analyzed by flow cytometry. Data were acquired as described above.