Isolation of Monocytes from Umbilical Cord Blood

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).