ABSTRACT: microRNAs are assumed to fine-tune cellular mRNA expression levels predisposing them for the control of cell development and cell fates. However, despite increasing appreciation of the role of miRNAs in controlling myeloid cell functions and some evidence for their contribution in in vitro monocyte differentiation, the in vivo role of specific miRNAs in the development and homeostasis of myeloid cells remains to be investigated. Here we profiled the microRNA expression of 8 different myeloid cell population including myeloid precursors (MP), myeloid dendritic cell precursors (MDP), common dendritic cell precursors (CDP), plasmacytoid dendritic cells (PDC) and Gr1+ monocytes from the bone marrow and three different spenic denritic cells (DCs): CD4+ DCs, CD8+ DCs and CD4-CD8- DCs. The source of the cells were female C57BL/6 animals in an age of 6 weeks. The FACS isolation was done on a pooled cell suspension from a minimum of 5 animals. C57BL/6 mice in an age of 6 weeks were purchased from Harlan. For bone marrow (BM) precursor isolation, ACK (0.15M NH4Cl, 0.1M KHCO3, 1mM EDTA in PBS) lyzed BM cells from femur and tibia were pooled from 15 mice and enriched by MACS with biotinylated CD135 (A2F10) followed by anti-biotin MACS beads (Miltenyi). The enriched fraction was further stained with Streptavidin-PerCP, CD117 (2B8), CD115 (AFS98) and lineage antibody cocktail (CD11b (M1/70), CD3 (145-2C11), CD4 (GK1.5), CD8M-NM-1 (53-6.7), Gr1 (RB6-8C5), Sca-1 (D7), B220 (RA3-6B2), Ter-119, CD11c (N418), NK1.1 (PK136)). Splenic dendritic cells were pre-enriched from 8 mice by CD11c MACS beads (Miltenyi) and further stained for CD8M-NM-1, CD4, CD11c and CD86 (PO3). BM plasmacytoid DCs were isolated from 5 mice, followed by Ficoll enrichment and stained for CD11c, CD317 (927) and Siglec H (eBio440c). BM monocytes were isolated from Ficoll enriched BM cells from 5 mice. Staining markers were: CD11b, Gr1 and CD115. All antibodies were purchased from BioLegend or eBioscience if not indicated otherwise. A FACSAria cytometer (Becton-Dickinson) was used for sorting and duplets were excluded by their FSC-H vs. FSC-W appearance.
