Relaxin increases circulating BMDECs in mice by colony assay and flow cytometry. (A) BMDEC-CFU stimulated by relaxin have characteristics of late outgrowth colonies. BMDEC-CFU are counted after 5 days of culture, and a brightfield view of colonies is shown (original magnification ×100). (B) True BMDEC-CFUs demonstrate Ulex europaeus 1 staining (original magnification ×200). (C) DiI-AcLDL uptake (original magnification ×200). (D) Dual staining in a merged image of Ulex and DiI-AcLDL staining (original magnification ×200). Instead of staining, the BMDEC-CFU cells can be propagated for months. (E) Brightfield view of cells after 3 months of propagation taking on a cobblestone, endothelial, monolayer appearance. (F) Brightfield view of propagated cells that were trypsinized and plated onto a coverslip for immunostaining with anti–VWF and/or anti–MECA-32. (G) Merged image of cells in panel F stained with anti-VWF (red) and the nuclear stain DAPI (blue; original magnification ×400). (H) Merged image of another coverslip stained with anti-VWF (red), anti–MECA-32 (green), and the nuclear stain DAPI (blue; original magnification ×630). (I-K) Mice were implanted with a osmotic pumps pump containing vehicle (I-J) or rhRLX (K-L), and after 5 days the peripheral blood was collected and stained with fluorochrome-conjugated monoclonal antibody to mouse endothelial cell markers Lin, Sca1, cKit, and Flk1. The gated cells were analyzed for Sca-1 and Lin characteristics (I,K), and the subpopulation of Sca1⁺ cells was analyzed for Flk1 and cKit expression (J,L). Background staining was corrected by use of isotype controls for all markers. Percentages shown are percent of gated cells, not total lymphocytes.