Characterization of confluent EPC cultures expanded from BM expressing KDR, VE-cadherin, CD133, and vWF. (A) Colony-forming and outgrowing endothelial cells from a representative patient are shown. A colony-forming unit expressing: (1) KDR (red), (2) VE-cadherin (green), and (3) merge shows independent cellular distribution of KDR and VE-cadherin. Outgrowing EPCs expressing: (4) KDR (green), (5) early hematopoietic antigen CD133 (red), and (6) merge show independent cellular localization of KDR and CD133. Cultures were maintained on 96-well, laminin-coated plates, where colony growth and confluent EPCs were observed after 14 and 18 days of culture, respectively. Indirect immunofluorescence was done with indicated antibodies, and appropriate isotype-matched serum with each of the primary antibodies was used as negative control (not shown). Images of the stained cells were digitally recorded on a confocal laser scanning microscope (Bio-Rad MRC 1024ES; Bio-Rad, Hercules, CA) and were generated at the projections of the z-stacks at 1024 × 1024 pixels. (B) Co-staining of EPCs grown from BM for expression of vWF (green), CD38 (red), and nuclear counterstain TO-PRO-3 (blue). Data from a representative patient show that EPCs are vWF-positive. Boxes on the left indicate patterns of immunocytochemical staining of the same EPCs; 3-D histogram on the right shows 2-color FACS analysis using anti-vWF-FITC, anti-CD38-PE, and isotype-specific control antibodies. (C) Two-color dot plot of primary EPC culture from a representative patient using anti-vWF-FITC, anti-CD133-PE, and anti-CD45-PE/Cy5 antibodies. Quadrants were set based upon isotype-specific controls for FITC and PE (and PE/Cy5, not shown). Numbers shown in the quadrants reflect percent of EPCs from which small, agranular debris was gated out based on forward and side scatter plots.