Characterization of isolated normal endothelial cells (NEC) from mice.

<p>(A) Representative flow cytometric analysis of NEC showing the expression of the endothelial markers CD31, CD105, CD144, and BS1-B4 lectin (mouse endothelial marker) (white area). Control levels with normal isotype IgG are shown in the gray area. Binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 revealed the high purity of the isolated NEC by FACS. (B) RT-PCR revealed that NEC expressed CD31, CD105, CD144, vascular endothelial growth factor receptor-1, 2 (VEGFR1 and VEGFR2) and not the monocyte marker CD11b and the hematopoietic marker CD45. To demonstrate that NEC were purified without any blood cell contamination, we also performed RT-PCR in mouse peripheral blood mononuclear cells (PBMC). Negative control was performed with template-free samples. (C) A375-SM cultured medium are analyzed by flow cytometry. The figure shows the forward and side scatter plot of the medium. A375-SM cells were cultured for 48 h. (D) Isolated TMV were observed by electron scanning microscopy. For sample preparation, TMV were coated on a glass cover for 4 h. (E, F) Flow cytometric analysis of TMV showing expression of HLA and phosphatidylserine (white area). (G) Western blotting analysis of TMV showing expression of HLA and β-actin. Same amount of proteins were loaded. These results indicate that some TMV might be shedding microvesicles derived from tumor plasma membrane.