Diaminofluorescein diacetate

NO levels were measured using the fluorescent probe diaminofluorescein diacetate (Sigma).^{32 (link)} Glass-bottom 35-mm dishes were seeded with HCAECs. At confluency, the HCAECs were briefly washed with Dulbecco's phosphate-buffered saline and then exposed to diaminofluorescein diacetate (final concentration 5 µmol/L, premixed with fresh endothelial cell medium) for 10 minutes. The HCAECs were then rinsed and imaged under a fluorescence microscope. To stimulate NO production, the HCAECs were treated with PDGF (20 ng/mL)+glutamine (1 µmol/L). Continuous imaging was performed for a period of 10 minutes following stimulation. The amount of NO produced is expressed as fluorescence intensity normalized to that at baseline.

Vessel rings were opened longitudinally. Following equilibration at 37°C for 30 min in 48-well plates with Krebs solution (Sigma-Aldrich; Merck KGaA), rings were incubated in the presence or absence of MPs (2.5 mg/ml) from each group for 1 h at 37°C. Control groups were treated with an equal volume of RPMI-1640. Then, the rings were treated with diaminofluorescein diacetate (10 µM; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. Vascular endothelial growth factor (50 ng/ml; Sigma-Aldrich; Merck KGaA) was added as a positive control. Laser scanning confocal microscopy (magnification, ×200, emission, 515 nm; excitation, 495 nm) was used to obtain fluorescence images. Fluorescence intensity was assessed using ImageJ software (version 1.52; National Institutes of Health) as previously described (16 (link)). RPMI-1640 medium in the absence of MPs was set as a blank control.