Ix71 widefield microscope system

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.

Calcium flux studies were carried out by plating DIV120 astrocytes on glass bottom microwell dishes (MatTek, Germany) and allowed to reach 90% confluence. Next, cells were incubated in Tyrode’s solution (in mM: 129 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 25 HEPES, 30 Glucose, pH 7.4) supplemented with 5 μM Fluo-4 (ThermoFisher Scientific) and 0.02% Pluronic F-127 (Sigma) for 45 min at RT and 5% CO2 in the dark. Fluorescence microscopy was performed using an Olympus IX71 widefield microscope system (Olympus, Japan) with a ×20 objective, using time-series frames with an interval of 2 s, using Hamamatsu ORCA-Flash high speed camera (Hamamatsu Photonics, Belgium). For each biological replicate, 10–20 cells were measured. Traces in the graphs represent the normalized average fluorescence intensity change over time.