2.7. Nitric Oxide (NO) Assay

A Griess reaction was used to measure the NO concentration in the culture medium of RAW 264.7 macrophages. Briefly, 100 μL of the culture medium conditioned by RAW 264.7 cells treated with EV (with or without anti-miR-214) or 10 ng/mL LPS was mixed 1:1 with the Griess reagent (1% sulfanilamide, 0.1% N-1-naphthalenediamine dihydrochloride, and 2.5% H₃PO₄, Merk Life Sciences S.r.l., Milan, Italy) and transferred to 96-well plates. After 10 min of incubation at RT, the absorbance was measured at 540 nm with a microplate reader (BioTek, Winooski, VT, USA). A sodium nitrite (NaNO₂) standard curve was used to calculate the amount of nitrite in each sample. The results were normalized to the number of cells.