Dihydrorhodamine respiratory burst assay for diagnosis of Chronic Granulomatous Disease?

Dear Tu,

please note also that DHA is, like DHE, Luminol and H2DCF-DA, highly diffusable and can react with ROS everywhere in the cell. WIth this probe you can only detect overall ROS production in the cell. Moreover, DHE and DHA are often misinterpreted as mitochondrial ROS probes, but they only accumulate after being oxidized by ROS anywhere in the cell.

For a more structured way to measure ROS, I can recommend the following probes:

Isoluminol: For extracellular/phagosomal ROS

Amplex Red: For extracellular/phagosomal H2O2

5,6-Carboxy-DCF: Cytosolic ROS

MitoSOX: For mitochondrial matrix ROS

Experimental setups that commonly lead to misinterpretation of ROS measurements in phagocytic immune cells such as macrophages and neutrophils are:

usage of only one type of ROS probe without explanation of the rationale behind the choice, such as compartment or ROS subspecies specificity or no specificity at all (“total cellular ROS”);

usage of unspecific inhibitors of ROS production or usage of only globally working ROS scavengers;

no genetic evidence for the ROS source (especially in case of Nox enzymes);

inconsistent use of different stimuli and cell types for experiments in one study.

In consequence, the highly complex and structured production of ROS in neutrophils or macrophages is often reduced to the phrase “ROS levels in neutrophils/macrophages”, without regarding the ROS-inducing stimulus, ROS sources, ROS subspecies and their specificity and selectivity for their targets, the compartmentalization of ROS production and the corresponding ROS probes, scavengers and inhibitors to be used for proper analysis.

If you like, please have a look at our recent review.

Article 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).

We used the probes and scavengers described in this review also for neutrophils, PBMC and microglia.

If you have any further question please ask anytime.

All the best and stay healthy,

Marc

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