I have recently purchased DAF to use as a nitric oxide indicator for mouse splenocytes, as I hope to determine which cell populations within the spleen are producing NO in response to a compound I am using (by flow cytometry). However, using DAF I seem to get a lot of background in B220+ cells, and less NO+ cells in my treated samples. Any suggestions on what this might be or how to decrease background would be welcome.
DAF-FM, as you know, passively diffuses across cell membrane and gets deacetylated by intracellular esterases thus by fluorescing the intracellular nitrosants but the quantum yield is in the range of 0.01 to 0.90 only. It is essentially non fluorescent until it reacts with nitrosants including s-nitrosothiols to form fluorescent benzotriazole. The sensitivity of DAF-FM for nitrite is poor at pH7.4. I would advise you to wash the cells in buffer prior to incubation with optimum DAF-FM (2-5µM for ~250000 cells) for about 30 min. then, wash the cells twice with the buffer before you estimate the fluorescence. You also have to remember that DAF-FM is first dissolved in DMSO approximately 7-10mM as a stock solution. Standardize the concentration of DAF-FM that you add to the cells (keep in mind DMSO is toxic to the cells – as a result any concentration higher than needed would result in false positive). You also have to standardize the incubation time and remember to keep the cells light protected during incubation. Your background will increase if the cells exude NO. DAF-FM is great for screening intracellular NO once you standardize the conditions. Good Luck!
Hey Amalia
It's not possible.
You have to label the cells and quantify NO in flow cytometry right after.
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