I am trying to quantify the release of Nitric oxide to test the Immunogenicity of my antigen on U937 cells (Monocytes).
Based on the literature, I am supposed to convert 1x105 cells/ml to unstimulated macrophages using PMA and then, after 48h, add LPS (used as positive control) to see if my macrophages release Nitric Oxide, which is Quantified using Griess reagent.
For some reason, there is no quantifiable release of Nitric oxide.
Do you guys suggest another method that I should follow?
Hi, you can try 4,5-diaminofluorescein diacetate (DAF-2 DA), a fluorescent probe for direct detection of Nitric Oxide (NO). This can provide more sensitive and real-time measurements. Also, you can include a known NO donor, as a positive control to validate your detection method. Good Luck.
