Phenotypic characterization of EPCs. CD31-selected cbEPCs were evaluated at passage 6. HDMECs and HSVSMCs served as positive and negative controls, respectively. (A) Cytometric analysis of cultured cbEPCs for endothelial markers CD34, VEGF-R2, CD146, CD31, VWF, and CD105, the mesenchymal marker CD90, and hematopoietic/monocytic markers CD45 and CD14. Solid gray histograms represent cells stained with fluorescent antibodies. Isotype-matched controls are overlaid in a black line on each histogram. (B) RT-PCR analysis of cbEPCs for endothelial markers CD34, VEGF-R2, CD31, VE-cadherin, VWF, and eNOS (the lanes were rearranged from the same picture to match panel A). (C) Indirect immunofluorescence of cultured cbEPCs grown in confluent monolayer showing positive staining for CD31, VE-cadherin, and VWF. VECTASHIELD mounting medium with 4,6 diamidino-2-phenylindole (DAPI; Vector Laboratories) was used. Images were taken with a Nikon Eclipse TE300 (Nikon, Melville, NY) using Spot Advance 3.5.9 software (Diagnostic Instruments) and a 20×/0.45 objective lens. Images were assembled into multipanel figures using Adobe Illustrator CS 11.0.0. (D) Up-regulation of E-selectin, ICAM-1, and VCAM-1 in cultured cbEPCs in response to TNF-α. Solid gray histograms represent cells stained with fluorescent antibodies, while black lines correspond to the isotype-matched control fluorescent antibodies.