I incubate cells with nanoparticles and I know that ROS are generated. But I don't know where they exactly appear: in the cells or outside the cells. Therefore I am searching for a fluorescent dye that can measure ROS production outside cells, ergo does not enter the cells. Does anyone know a possibility to measure the ROS production outside cells or do I have to first incubate the cells with the media and then remove it and measure without the cells? Which dye would you recommend in a cell-free system?
Molecular Probes (Thermo-Fisher) has a product called OxyBurst Green for this purpose.
Here is the chapter of the catalog on fluorescent dyes for ROS purposes:
https://www.thermofisher.com/content/dam/LifeTech/global/technical-reference-library/Molecular%20Probes%20Handbook/chapter-pdfs/Ch-18-Reactive-Oxygen-Species.pdf?icid=WE216841
I think you can estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK).
Concerning the OxyBurst Green I learnt that although it was originally stated that it measures extracellular ROS it is taken up into endosomes. Adam B Shapiro did you already work with the product?
Perhaps you can also look at using Lucigen (I think it measures extracellular ROS) through chemiluminesence
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