Streptavidin flowcomp dynabeads

A 20-gauge angiocatheter was inserted into a superficial forearm vein. Under sterile conditions, three J-shaped vascular guide wires (Arrow) were sequentially advanced into the vein up to 10 cm. ECs were retrieved from wire tips by washing with EC dissociation buffer. Harvesting yielded ∼2000 to 5000 ECs. For immunofluorescence, ECs were recovered by centrifugation at 4°C, 150g for 6 min, and then the cell pellet was resuspended in red blood cell (RBC) lysis buffer (eBioscience), incubated at 4°C for 5 min, centrifuged at 150g for 6 min, fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) in phosphate-buffered saline (PBS) for 10 min, washed twice with PBS, transferred to poly-l-lysine–coated slides (Sigma), and then air-dried at 37°C. The slides were stored at −80°C until analyzed. For phage display and mRNA extraction, the cell pellet was resuspended in isolation buffer, incubated with biotinylated mouse anti-human monoclonal antibody directed against CD146 (1:200; catalog no. MAB16985B, Millipore) at 4°C for 15 min, incubated at 4°C with Streptavidin FlowComp Dynabeads (1:100; Invitrogen) for 45 min, and then subjected to EC isolation by magnet. For flow cytometry, the cell pellet was resuspended in RBC lysis buffer, incubated at 4°C for 5 min, and then centrifuged at 150g for 6 min. The isolated cell pellet was resuspended in PBS.