DAF-FM DA

DAF-FM DA (Diaminofluorescein-FM diacetate) is a fluorescent probe for the detection and bioimaging of nitric oxide (NO). DAF-FM DA spontaneously crosses the plasma membrane and is subsequently cleaved by esterases to generate intracellular DAF-FM (Ex/Em=495/515 nm)^[1]. In Vitro:Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1.1 Preparation of stock solution^[2]
Use anhydrous DMSO to prepare a 5 mM DAF-FM DA stock solution.
Note: It is recommended that the DAF-FM DA stock solution be stored at -20℃ or -80℃ in the dark after aliquoting.
1.2 Preparation of working solution
Dilute the stock solution with Hank’s buffer or serum-free medium to prepare a 5 μM DAF-FM DA working solution.
Note: Please adjust the concentration of DAF-FM DA working solution according to actual conditions and prepare it before use.
2 Staining steps
1) Seed breast cancer cells in a 6-well plate.
2) Aspirate the culture medium.
3) Add 5 μM DAF-FM DA working solution to each well and incubate at 37°C in the dark for 30 min to allow the probe to enter the cells.
4) Replace with fresh Hank’s buffer and continue incubation for 20 min to complete the deesterification reaction.
5) Counterstain the cell nucleus with Hoechst dye.
6) Quantify the fluorescence intensity using a microplate reader at 488 nm excitation and 515 nm emission, and normalize to the number of cells stained with Hoechst.
In Vivo:Using sections of zebrafish embryos live-stained with DAF-FM DA (5 μM), could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation^[1].