Diamond pdc isolation kit

All human studies were performed according to the investigator's protocol approved by the Institutional Review Board of Columbia University. PBMC were isolated from healthy adult volunteers by Histopaque density gradient centrifugation; where indicated, pDCs were enriched using the Diamond pDC isolation kit (Miltenyi Biotec) to >95% purity. Human PBMC subsets for expression analysis were purchased from Allcells.
For pDC activation, PBMCs were plated at 5 × 3 10⁵ /well of flat-bottom 96 well plates in RPMI medium with 10% FCS in the presence of affinity-purified polyclonal goat IgG antibody to the extracellular domain of human PTPRS (R&D Systems) or of control goat IgG (Santa Cruz Biotechnology). In titration experiments, antibody concentrations >0.01 μg/mL were found to have the same effect. CpG type A (ODN 2216, Invivogen) was added 1 hr later at 5 mM concentration. After 6 hr, protein transport inhibitor (BD Golgi Plug™, BD Biosciences) was added and cells were incubated for additional 10 hr. For the analysis by immunofluorescence, purified pDC were pre-incubated with anti-PTPRS or control IgG and then activated with CpG for 3 hr.

Peripheral blood mononuclear cells were obtained from the buffy coat of healthy donors (provided by the Blood Transfusion Center of S. Orsola-Malpighi Hospital, Bologna, Italy) and separated by Ficoll gradient density centrifugation using Lympholyte-H. (Cedarlane Laboratories, Euroclone, Pero (MI), Italy). PDCs were isolated using the Diamond pDC isolation kit (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. For determination of purity, a fraction of isolated pDCs, stained for CD303 (BDCA-2)-APC and CD123-PE was analyzed by flow cytometry. The purity of pDCs isolated from the samples was >95%.