Hello everyone and thanks in advance for taking the time to read through my question!

I am doing some Seahorse experiments on macrophages (not an immortalized cell line, in case that it makes any difference), particularly the Cell Mito Stress test.

I am also starting to measure ROS production using DHR, but I am struggling to understand the molecular differences between the two.

I contacted Agilent with the question and their response was "Seahorse measures changes of oxygen consumption rate and extracellular acidification in response to a series of mitochondrial stress compounds. Mito Stress Test does not directly detect reactive oxygen species."

The thing is (and here's where my doubt comes from), that in their explanatory video show how the first fluorophore in the plate receptor detects Oxygen, and the second is sensitive to protons. Which makes me think that the first fluorophore does react to the same oxygen that could be detected using DHR?

If I understood properly though, Seahorse detects these variations extracellularly while DHR works intracellularly, that's why I suspect they are measuring two different things that I am not quite understanding?

Thank you so very much in advance for your help!

Best,

Alba