Analysis of nitrite oxide

Nitric oxide production was analyzed using Griess Reagent (cat # G4410-10G, Sigma) using procedure published elsewhere with slight modifications⁸². Briefly, cell media collected from BV2 cells or primary microglia was centrifuged at 500 g for 2 mins to remove cell debris if any, then 70 µl of supernatant was added to 70 µl Griess reagent in a clean 96-well plates (Cell Star, Greiner Bio-One, cat #655180), incubated for 5 min in darkness at room temperature, and absorbance was recorded at 548 nm and 595 nm using iMark microplate reader (cat #1681135, Bio-Rad). All samples were analyzed in triplicates. As control for absorbance by media components, 70 µl of fresh DMEM media (10% HS, 1X antibiotics) was mixed and incubated with 70 µl Griess reagent in parallel. The amounts of nitrite oxide was quantified using a calibration curve build using freshly prepared sodium nitrite (Sigma, cat #237213) and Griess reagent.