2.8. Nitric oxide measurement

In 12-well plates, intracellular nitric oxide (NO) assessments were made by flow cytometry. HUVECs were incubated for 30 min in complete endothelial cell media with 5 μM of 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM) and 100 μM ATP at 37 °C in 5% CO2. Cells were washed with PBS and then incubated with fresh endothelial cell media for 15 min. Cells were collected in 0.25% trypsin-EDTA with complete medium and analyzed by flow cytometry using a Guava EasyCyte Mini and the Guava Express Plus program (Guava Technologies, City State). Acquisitions involved 5000 events.