*4.5. Nitrite Levels Quantitation Assay*

Griess reagent assay was performed to evaluate the influence of glucose or insulin on nitric oxide (NO) levels in neuron-like cells [60]. This spectrophotometric assay is based on the formation of an azo dye by the reaction of NO2 − present in the sample with the Griess reagent. For each od experimental setting, the 1 × 104 cells were seeded in a 96-well plate. After 1 h and 24 h incubation with a series of glucose or insulin concentrations, 50 μL of supernatant was transferred into new 96-well plates. The 1:1 mixture (*v/v*) reagent A (1% sulfanilamide in 5% phosphoric acid) and reagent B (0.1% N-(1- Naphthyl) ethylenediamine dihydrochloride) was added to supernatants and left for 20 min in the dark at room temperature. After incubation, the absorbance at 548 nm was measured using a Varioskan LUX microplate reader (Thermo Scientific, Waltham, MA, USA).
