Cd31 bv605

AGM (aorta-gonad-mesonephros) regions (including trunk portion of the vitelline and umbilical arteries) of E10/11 embryos were dissected as previously described (Medvinsky et al., 2008 ). Single cell suspensions were prepared by enzymatically digesting tissues at 37°C in 0.125% type 1 collagenase (C0130, Sigma-Aldrich) for 45 min, after which tissues were further mechanically disrupted with a P1000 pipette. Cells were subsequently passed over a 0.35 μm strainer, collagenase washed away with PBS+10%FBS+1%P/S (PFP), spun down (234g) and resuspended in PFP for flow cytometric antibody staining. Cells were incubated at 4°C for 30 min in PFP with directly conjugated antibodies against CD31 (CD31-BV605, 1:100, cat# 102427, Biolegend), cKit (cKit-BV421, 1:200, cat.# 562609, BD) and CD27 (CD27-APC, 1:75, cat.# 17-0271-82. eBioscience). Excess of antibodies was washed away with PFP before fluorescence activated cell sorting (FACS) on an AriaII (BD). Dead cell exclusion was by Hoechst 33342 and gates were set based on unstained WT and fluorescence-minus-one (FMO) controls. Sorted single cells were collected in 50% FBS/PBS for functional analyses (transplantation + bulk CFU-C assay). Both collagenase and FCS were batch tested for optimal AGM conditions.