I´m sorry, I cannot answer your question, but I can recommend to you to investigate the HyPerRed-mito plasmid and see if there is an equivalent for phagolysosomes to investigate H2O2 levels.

For lysozyme, I´m afraid that you will need to do a subcellular fractionation or inmunofluorecence.

Thanks for your reply. Pablo Veloso

Hi,

The concentration of H2O2 in the phagolysosome depends on your stimulus (the phagocytosed particle). Without phagocytosis, you will have only a low amount of background H2O2 production.

So, if you want to analyze the phagolysosomes in your THP-1 cells, you need a particle e.g. latex beads or better zymosan particles, or some microorganisms like bacteria or fungi, which should be phagocytosed by your cells.

For simplicity, I would use zymosan particles, which in contrast to latex beads should activate some pro-inflammatory responses in your THP-1 cells, including production of H2O2.

In order to analyze the production of ROS e.g. H2O2 in a specific cellular compartment such as the phagolysosome, it is important to choose an adequate, non-diffusable ROS probe. In the ideal case, you should couple your ROS probe directly onto your particle of choice. The probe will start to fluoresce upon oxidation inside the phagolysosome and you can detect and quantify the fluorescence signal in a plate reader.

For further information, or technical details about ROS measurements, I would like to highly recommend our paper, which focuses on ROS measurements in different cellular compartments of phagocytic cells:

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling

https://stke.sciencemag.org/content/12/568/eaar5926

For theoretical background, I would like to recommend an excellent review "Functions of ROS in macrophages and antimicrobial immunity" by my colleague Marc Herb. Here he gives an overview about ROS and their different sources in macrophages, summarizes the roles of ROS in antimicrobial immune defense and provides an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers.

If you like, please have a look at:

Functions of ROS in Macrophages and Antimicrobial Immunity February 2021, Antioxidants 10(2):313 DOI: https://doi.org/10.3390/antiox10020313

Kind regards,

Alex

Alexander Gluschko thanks for the wonderful information given, Thanks alot.