I am doing the ROS production of BMDM from B6 mice, I usually plant the macrophages in 1*105 each well, wash with warm HBSS once and add 200ul HBSS with luminol (100uM) and 5U HRP each well, incubate for 20 min in 37 Degrees Celsius, measure (2300 EnSpire Multilabel Plate Reader) for 20-60min, and then add stimulation: PMA(800nM/400nM), fMLP(100uM/10uM), same volume of DMSO as negative control, measure for another 60min.
My data showed the RLU increased stable before adding the stimulation, but all the data decline dramatically after I added stimulation and DMSO, and increased slightly since then. It seems the PMA, fMLP did not work at all, the curve of PMA and fMLP consist with the DMSO.
I have seen lots of literature about this experiment, their data all show dramatically increase of ROS production after stimulation, why my data shows nothing significant? The PMA and fMLP are new, and they did work in neutrophils, I wonder why they do not work in macrophages? Is there something I can improve to get the result?
Did you check the viability of your BMDM cells? It's possible that they have not survived the isolation procedure.
Dear Paige,
Thank you for your answer, I checked the status of my BMDM before stimulation, and they were in good condition, I considered that maybe I add too much DMSO to them. I have corrected the concentration of DMSO by diluted the PMA and fMLP in 20ul HBSS, and then added to the BMDM, but it still not work. However, this method worked in fMLP stimulation on Neutrophils, and unstable in PMA. I am still trying to figure out what I can improve.
In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
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
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages.
If you like, please have a look at:
Article Macrophages target Listeria monocytogenes by two discrete no...
All the best and stay healthy,
Marc
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