Intracellular Nitric Oxide Quantification

We assessed intracellular NO with DAF-2DA, a diaminofluorescein-2 diacetate fluorescence probe (10 μM). DIV120 astrocytes grown to 90% confluence were incubated with NMM containing DAF-2AM (ThermoFisher Scientific) for 60 min at 37 °C followed by a 30-min post-incubation period in NMM without the fluorescent indicator. NO production analysis was carried out using an Olympus IX71 widefield microscope system (Olympus) with excitation and emission wavelengths of 460–495 nm and 510–550 nm, respectively^{10 (link)}. We recorded images using imageJ’s micromanager- Hamamatsu ORCA-Flash high speed camera (Hamamatsu Photonics) and measured the increases in the green fluorescence intensity.