Could you kindly inform what are the best concentrations of 2',7'-dichlorofluorescein diacetate stock (in DMSO) and working solutions (in PBS) for detecting ROS generation in leukocytes by
flow cytometry?
Are there any staining protocols?
Thank you all in advance.
Hey Anton,
Our stock solution for DCF is 10 mM in DMSO. We resuspend our cells (peritoneal macrophages, bone marrow-derived macropahges, PBMCs) in HBSS with a f.c. of 20 µM and incubate for 15 min at 37°C. Then we wash one time with HBSS, resupend in FACS buffer (PBS + 0,5 % BSA and 2mM EDTA), transfer the solution to a FACS tube and measure at the flow cytometer.
I also want to add that 2',7'-dichlorofluorescein diacetate (which nearly everybody uses) diffuses over membranes (so into organelles and outside the cell) and therefore gives you only an "overall" ROS production that diminshs fast.
We use a derivate 5(6)-dicarboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester). We tested the two DCF derivates and the latter has a much better retention in the cell and did for example not diffuse into mitochondria. So with this derivate you only measure cytosolic ROS. This was also tested by others:
S. L. Hempel, G. R. Buettner, Y. Q. O’Malley, D. A. Wessels, D. M. Flaherty,
Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: Comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-
dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol. Med. 27, 146–159 (1999).
Please feel free to ask any further question.
All the best,
Marc
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
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