Cd14 magnetic bead isolation

Umbilical cord blood was collected from enrolled infants immediately after the delivery of the placenta. The umbilical cord blood was stored in the University of Michigan blood bank at 4^oC and the samples were retrieved from the blood bank within 5 days of collection as previously described[17 (link)]. The blood samples were diluted 1:2 with sterile 0.9% saline and Ficoll-Isopaque density gradient centrifugation was used to isolate mononuclear cells. Mononuclear cells underwent CD14+ magnetic bead isolation according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA, 130–050-201). The average purity of the monocytes was 92% by flow cytometry using anti-human CD14 (BioLegend, San Diego, CA, USA, clone HCD14, PerCP/cy5.5). Isolated CD14+ cells were stored in recovery cell culture freezing media (Life Technologies, Carlsbad, CA, USA) in cryovials at −80^oC until use. Monocyte viability post freezing was 70–75% by flow cytometry using DAPI (Molecular probes, Eugene, OR).

Human PBMCs were obtained by gradient centrifugation of leukapheresis collars from platelet donors. PBMCs (4 × 10⁶) were plated with 10⁵ irradiated K562-WT or K562-GM-CSF–secreting cells. On days 4 to 6 of culture, floating cells were harvested and adherent cells were detached with 2 mmol/L EDTA. Cells were stained for flow cytometry in the presence of 1 mmol/L EDTA. Dead cells were discriminated by using the Live/Dead Fixable dyes from Invitrogen. For monocyte produced cytokines, 10⁶ monocytes from PBMCs (CD14⁺ magnetic bead isolation, Miltenyi Biotec) were plated with 10⁵ irradiated K562-WT or K562-GM-CSF–secreting cells. Human and mouse CCL22 and CCL17 levels were measured using ELISAs (R&D Systems). FoxP3 expression was detected by using anti-FoxP3 (clone 236A/E7). For Treg suppression assays, CD25^hi cells were collected by flow sorting T cells obtained by negative selection (Miltenyi) and incubated with CD8⁺ or CD4⁺ T cells cultured with activating beads (Miltenyi, 130–092-909), according to the manufacturer’s recommendations.