Mv bullet kit

Normal human lymphatic microvascular endothelial cells (HMVEC‐dLy) (Lonza, MD) referred in the text as HLEC were grown as tissue culture monolayers in EGM™‐2 medium supplemented with MV BulletKit (Lonza). For tube formation assays, three‐dimensional cultures were prepared with 2.5 × 10⁵ cells seeded in MW6 plates covered by a layer of Matrigel (BD, NJ). When indicated, cells were pretreated for 12 h with 0.5 µg/ml BO‐110 or vehicle control. Treatment was maintained thereafter. Pictures were acquired 10 h after seeding the cells. Time‐lapse videos were acquired in the same experimental conditions using a Leica Thunder widefield microscope with a 10× objective 0.45NA and LASX v3.7 acquisition software. Images were captured every 45 min. Videos were processed using Fiji (ImageJ) software. For fluorescence detection of HLEC in this time‐lapse imaging, cells were labeled with CellTracker™ Green CMFDA Dye (Thermo Fisher, C7025, Waltham, MA) following the manufacturer’s instructions.

EPCs were isolated from 8-week-old male C57 mice. In brief, whole BM cells were collected under sterile conditions from femur (and tibias) by flushing the shaft with phosphate-buffered saline (PBS). The mononuclear cells were isolated by percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA) at 2,000 g for 20 minutes. After three rinses, the isolated cells were cultured in endothelial basal medium-2 (EBM-2) with an MV Bullet Kit (Lonza, Walkersville, MD, USA). After 7 days of culture, the EPC markers DiI-labeled acetylated low-density lipoprotein (DiI-AcLDL) (Sigma-Aldrich) and bandeiraea simplicifolia lectin 1 (BS-1 lectin) (Sigma-Aldrich) were confirmed by immunofluorescence analysis.

Human coronary artery endothelial cells (HCAEC) and human endocardial microvascular endothelial cells (HMVEC) were obtained from Lonza (Walkersville, MD) and were grown in EGM-2 supplemented with MV Bullet Kit. To expose HCAECs to hypoxia-reoxygenation (H/R), HCAECs were grown to confluence and then incubated with deoxygenated (flushed with 95% N₂/5% CO₂)-EGM-2 in Billups-Rothenberg modular chambers at 37 °C in 95% N₂/5% CO₂ gas mixture for an indicated period followed by reoxygenation in 5% CO₂/95% air. To obtain preconditioned medium (PM), HMVECs were incubated with deoxygenated-EGM-2 without growth supplements for 1 hour at 37 °C in Billups-Rothenberg modular chambers flushed with a 95% N₂/5% CO₂ gas mixture. After 1 hour of hypoxia, the cells were reoxygenated in normoxic conditions. At this point, medium from HMVECs was collected and used as PM to treat HCAECs. Cardiomyocytes and endothelial cells from adult mouse hearts were isolated by Liberase TH (Roche Applied Science) and collagenase II digestion, respectively.^{33 (link)}