Human umbilical vein endothelial cells (HUVEC) (Lot#0000120825; Lonza®, Walkersville, MD, USA) were cultured at 37 °C and 5% CO2 in endothelial basal media (EBM-2) supplemented with a bullet kit (Lonza®) containing human fibroblast growth factor B, hydrocortisone, vascular endothelial growth factor, ascorbic acid, heparin, human

Rapamycin in vivo endothelial growth factor, and fetal bovine serum. For cell passage, cultures were incubated to approximately 40% confluence within the culture flask, according to LONZA guidelines. For experiments, cultures were incubated to approximately 50% confluence then harvested by exposure to trypsin–EDTA (Lonza®) for 2 min at 37 °C. Cell suspensions were centrifuged at 201g for 5 min in a 5810R tabletop centrifuge (Eppendorf,

Westbury, NY, USA), and resuspended in endothelial growth media at a concentration of 1.0 × 106 cells/mL in 1 mL aliquots maintained in 12 × 75 mm round bottom plastic tubes (VWR, Edmonton Canada) prior to experimentation. The dual fluorescent assay (SytoEB) Bcl-2 inhibitor uses a combination of two fluorescent dyes, Syto13 (Molecular Probes, Eugene, OR, USA) and ethidium bromide (EB) (Sigma–Aldrich, Mississauga, ON, Canada) to assess cell membrane integrity. Syto13 is a DNA/RNA binding stain that permeates all cells and fluoresces green on excitation by UV wavelengths. Ethidium bromide permeates cells with damaged plasma membranes, exhibiting red fluorescence upon UV exposure. The combination of these two dyes makes a binary assay with membrane intact cells exhibiting green fluorescence (Syto13) and membrane compromised cells exhibiting red fluorescence (EB). The SytoEB stain was prepared using 1× phosphate buffered saline (PBS), and aliquots of Syto and EB diluted from the stock solution. The final dye was comprised of 25 μM EB and 12.5 μM Syto13. 10 μL of the prepared dye were added to the 1 mL aliquot of HUVEC in suspension and incubated for 2 min at room temperature before analysis. The ratiometric dye 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine-iodide

(JC-1) (Molecular Probes, Eugene, OR, USA) was used as an indicator of mitochondrial membrane potential before of HUVEC in suspension. The fluorescence shifts from green (∼525 nm) in low polarization states (non-functional mitochondria) to red (∼590 nm) in high polarization states (functioning mitochondria). This change in color of fluorescence is based on a concentration-dependent shift from monomers of the dye which fluoresce green to J-aggregates which fluoresce red [34]. Initially the dye is present as cationic monomers (green) that permeate into cells, influenced by the negative intracellular potential. In healthy cells these monomers permeate into the mitochondrial matrix, drawn by the electronegative interior of mitochondria where these monomers form J-aggregates (red) [15].