Ficoll hystopaque plus

Bone marrow (BM)-derived CD34+ were obtained from Calabretta’s lab at Thomas Jefferson University. CD34+ CD33− cells were sorted at the Flow Cytometry facility at Wistar Institute. Fetal liver (FL)-derived CD34+ cells were obtained from Stem Cell and Xenograft Core at University of Pennsylvania and were maintained in StemSpan SFEM medium supplemented with CC100 cytokine cocktail (Stem Cell Technologies). De-identified human cord blood (CB) was obtained from volunteers with informed consent at Helen F. Graham Cancer Center and Research Institute at Christiana Hospital. Mononucleated cells (MNC) were separated with Ficoll-Hystopaque Plus (GE Healthcare). CD34+ cells were then isolated using human CD34 MicroBeads Kit (Miltenyi Biotec) following manufacturer’s instructions. CD34+ were maintained in StemSpan SFEM medium supplemented with 1X CC100 cytokine cocktail. BM- and FL-derived CD34+ were used for CFU assay, BM-, FL- and CB-derived CD34+ were in vitro differentiated and used for RNA-seq and ChIP-seq experiments, respectively. Circulating monocytes were obtained from the Human Immunology Core at University of Pennsylvania.