Nitric Oxide Assay and Quantification

Endothelial cell function was assessed via quantification of nitric oxide production using the fluorescent probe, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate (Thermo Fisher Scientific).32 Samples were incubated with 5 μM solution of DAF-FM in DMSO for 40 min at 37 °C and then washed in PBS to remove excess probe. After an additional 30 min of incubation in EGM-2MV endothelial growth medium (Lonza) to allow for complete de-esterification of intracellular diacetates, the living cells were then imaged using a confocal microscope (Zeiss LSM710). 3D z-stacks were acquired, with a z-stack thickness of 300–400 µm, and a 1 µm optical section thickness. DAF-FM intensity was captured and quantified using ImageJ by thresholding image stacks based on hue, saturation and pixel value. The measurement of DAF-FM integrated density was extracted, and the values were normalized by total cell number based on Hoechst staining. For each sample, 5 images at 10× magnification were analyzed (n = 3).