DAF-2 DA (5,6-Diaminofluorescein diacetate) is a cell-permeable fluorescent probe used for detecting nitric oxide (NO), requiring use in a serum-free environment free from phenol red interference. It has an excitation wavelength of 491 nm and an emission wavelength of 513 nm, with a commonly used working concentration of 1–10 μm.

Steps for assessing total mitochondrial nitric oxide levels:
1. SH-SY5Y cells were seeded in duplicate at a density of 5 × 10^3 cells/well in black 96-well cell culture plates.
2. After gelsemium treatment, cells were incubated with DAF-2 DA (10 μM, 15 minutes) on an orbital shaker at room temperature in the dark.
3. After washing twice with HBSS (Sigma) using a Cytation 3 cell imaging multi-function microplate reader, the formation of a green fluorescent product was detected at 485 nm (excitation)/538 nm (emission). The fluorescence intensity is the total cellular nitric oxide level. [2]