Hcd33 pe

Peripheral blood and bone marrow cells from xenografts were processed as previously described (10 (link)). V-AML was isolated from bones that were crushed with a mortar and pestle and collagenased as described by Hooper et al (30 (link)). Non-parenchymal cells from the liver of chimeric mice were generated essentially as described (31 (link)), following a single collagenase digestion. Antibodies were purchased from BD Biosciences unless otherwise indicated. For human cell engraftment, mCD45.1-PE-Cy7 (eBioscience), hCD13-PE, hCD33-PE, hCD45-APC, hCD3-FITC were used and cells were analyzed with a BD FACSCalibur or a BD LSRII flow cytometer. Following staining with mCD45.1-FITC, hCD33-PE, hCD13-PE, mCD31-APC, V-AML cells were then isolated by FACS sorting using a BD InFlux with a 150 micron nozzle. ECFCs were stained with hCD14-APC, hCD146-PE, hCD105-PE (Invitrogen) and hUEA-1-FITC (Sigma) and hCD45-PE, hCD115-PE, hCD144-PE, hVEGF-R2-PE (R&D Systems) and Dil-ac-LDL (BD Biosciences) uptake by incubating cells with 10 μg/ml Dilac-LDL in EGM-2 media for 4 hours at 37°C prior to analysis. Analysis was performed using a BD FACSCanto II flow cytometer. For all flow cytometry, dead cells were excluded using propidium iodide and scatter gates and doublets were excluded using the pulse width parameter.