DAF-FM DA assay

Intracellular NO was measured in real time using the NO-specific fluorescence probe DAF-FM DA solution (Sigma-Aldrich). DAF-FM DA diffuses freely across the membrane and is hydrolyzed by intracellular esterases, resulting in the formation of DAF-FM. Intracellular DAF-FM reacts with the NO oxidation product N₂O₂, which generates the stable highly fluorescent derivative DAF-FM triazole. Cells were washed with modified HEPES buffer (20 mM HEPES buffer [Gibco] with 5 mM glucose, 50 µM l-arginine, and 0.1% BSA, pH 7.0–7.4), incubated with 5 µM DAF-FM DA in modified HEPES buffer for 30 min at room temperature, washed again, and finally incubated in modified HEPES buffer for 30 min at 37°C in the absence or presence of 1 mM L-NMMA. Fluorescence (emission wavelength, 485 nm; excitation wavelength, 538 nm) was measured at 37°C from 1 to 10 min using a fluorescence microtiter plate reader (Synergy HTX Multi-Mode Reader, BioTek). eNOS activity was expressed as the VEGFA-dependent increase in fluorescence per microgram of cellular protein. To determine the cellular protein content, the same cells were lysed in 1% (vol/vol) Triton X-100 and analyzed for protein content with the BCA protein detection kit. DAF-FM DA experiments were repeated three times. Within each experiment, four wells were used for each NO measurement.