Cd14 specific immunobeads

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).

Monocytes were isolated from either normal human buffy coat PBMC (LeukoPaks; Central Blood Bank of Pittsburgh) using CD14-specific immunobeads (Miltenyi, San Diego, CA), or obtained as the elutriated monocyte fraction of normal human volunteer leukapheresis products (Institute for Transfusion Medicine, Pittsburgh, PA) under an IRB-approved protocol. Immature DC (imDC) were generated as described [20 (link)], except that those generated from elutriated monocytes were cultured in DC serum-free media (Corning) without antibiotics using GMP grade reagents. DCreg were generated by addition of VitD3 (20mM; Sigma, St. Louis, MO) on days 0 and 4 and rhIL-10 (60ng/ml; Peprotech, Rocky Hill, NJ) on day 4 of culture. Lipopolysaccharide (LPS; Salmonella minnesota R595; 0.5 µg/ml, Enzo Life Sciences, Farmingdale, NY) was added to DC cultures on day 6 to evaluate their response to a potent maturation-inducing agent. Responses to soluble CD40 ligand (sCD40L; MegaCD40L, 100ng/ml, Enzo Life Sciences) or a pro-inflammatory cytokine cocktail (PCC) consisting of IL-6, TNFα, IL-1β (all 10ng/ml; Peprotech) and prostaglandin (PG)E₂ (1µg/ml; Sigma) were also evaluated. Upon harvest, DC were washed thoroughly (1× PBS) before phenotypic and functional analyses.