DAF 2DA is most widely probe for NO measurement. In Vitro: Nitric oxide is a free radical signal molecule. Various methods are available for measurement of NO. Out of all methods, fluorescent probes to localize NO is very widely used method. Diaminofl uorescein in diacetate form (DAF 2DA) is most widely probe for NO measurement. This method is based on application of 4,5-diaminofl uorescein diacetate (DAF 2DA) which is actively diffused into cells, once taken up by cells cytoplasmic esterases cleave the acetate groups to generate 4,5-diaminofl uorescein; DAF-2. The generated DAF-2 can readily react with N₂O₃, which is an oxidation product of NO to generate the highly fluorescent DAF-2T (triazolofl uorescein).