Anti cd31 magnetic dynabeads

Primary carotid ECs for use as a reference group were isolated as described previously [27 (link)] from animals from which ECFCs were previously collected. This study includes case-matched ECs and ECFCs from two animals. Briefly, whole carotid arteries were rapidly excised from male juvenile Papio anubis baboons at the time of necropsy, and flushed with and stored in ice-cold Dulbecco’s phosphate buffered saline (PBS, 0.1M, Gibco) containing 2–4% penicillin-streptomycin (PS, Gibco) for 1–4 hours. To release ECs from the vessel wall, arteries were clamped shut at bottom, and filled with a 37°C solution of 600U/mL collagenase type II (Worthington Biochemical) dissolved in endothelial basal medium (EBM, Lonza) and incubated for five minutes. EC-containing collagenase solution was dripped directly into tissue culture wells which were precoated with 50 μg/mL collagen-I from rat tail (Corning) and filled with EGM-2 supplemented with 18% FBS. Cultures were fed every other day with EGM-2 + 18% FBS for up to four weeks until EC colonies occupying at least half of the well were seen. These colonies were passaged using TrypLE, sorted using anti-CD31 magnetic dynabeads (Invitrogen), and expanded in T-150 flasks. Between isolation and experiments, cells were maintained in cryogenic storage.

ECFCs were isolated from the blood of male juvenile Papio anubis baboons using a previously established protocol [26 (link)]. Briefly, 50 mL of venous blood was drawn into a 7% ACD solution. After no more than 45 minutes, blood was mixed 1:1 with Hank’s Buffered Saline Solution (HBSS, Corning) and carefully layered on top of Histopaque-1077 (Sigma). The tubes were then centrifuged (800 G, 30 minutes, without braking) to isolate the buffy coat containing mononuclear cells. This mononuclear cell suspension was isolated, washed and plated on tissue culture plastic pre-coated with 50μg/mL fibronectin (Sigma). Cultures were fed daily for the first seven days of culture, and every other day for up to four additional weeks with EGM-2 (Lonza) + 18% fetal bovine serum (FBS, Hyclone). When colonies of cobblestone-shaped cells appeared, they were isolated by manually scraping the non-colony-occupied portions of wells and passaging with TrypLE (Gibco). These cells were expanded in T-25 flasks as described below, and upon reaching confluence, were passaged, sorted using anti-CD31 magnetic dynabeads (Invitrogen), and expanded in T-150 flasks, as described below.