The isolation of human monocytes and dendritic cells from de-identified human subjects was performed under IRB Protocol #2015-2437 and through the UC Irvine ICTS Blood Donor Program. Donor sex: CD14 M = 2 males, 3 females. CD16 M = 2 males, 2 females. Blood DCs were all from female donors. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll-paque (GE Healthcare) gradient separation. In brief, blood was layered on top of Ficoll-Paque and centrifuged in swinging bucket rotator without brake (400 × g, 40 minutes, 18°C). After centrifugation, plasma and upper layers were removed and PBMCs isolated from the interphase. Cells were then washed once with ice-cold PBS and used immediately. CD14 and CD16 monocytes were isolated via negative selection from PBMCs using the EasySep™ Monocyte Enrichment Kit (Stemcell Technologies) per manufacturer’s instructions. Blood dendritic cells were isolated via negative selection from PBMCs using the EasySep™ Human Pan-DC Pre-Enrichment Kit (Stemcell Technologies) per manufacturer’s instructions. Isolated cells were washed three times with PBS and sorted by FACS for either RNA-sequence analysis or used for further macrophage differentiation.
