Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

Abstract

Background: Recently, new "targeted" fluorescent probes that react selectively with reactive oxygen and nitrogen species to yield specific products have been discovered. Results: High-throughput fluorescence and HPLC-based methodology for global profiling of ROS/RNS is described. Conclusion: This methodology enables real-time monitoring of multiple oxidants in cellular systems. Significance: The global profiling approach using different ROS/RNS-specific fluorescent probes will help establish the identity of oxidants in redox regulation and signaling.

Reliability of ROS and RNS detection in hematopoietic stem cells − potential issues with probes and target cell population

“…This means that oxidant-sensitive probes are generally non-specific for a particular oxidant, and their complex chemical behavior makes them unsuitable for mechanistic studies of cellular events that are induced by specific ROS and RNS species (Wardman, 2007;Winterbourn, 2014). Owing to the fact that a positive signal can be generated from non-specific reactions, the reactivity of oxidant-sensitive probes with particular ROS or RNS species should be confirmed by other techniques or by use of appropriate inhibitors (Zielonka et al, 2012b). However, their main advantage is that they are easy to use.…”

“…However, under conditions that generate both NO• and O 2 • − , it is not a suitable method to measure NO• selectively, because O 2 • − contributes to the oxidation of DAF-2 into fluorescent DFA-2T. Therefore, NOS inhibitors or ROS-scavengers should be included in the assay in order to discriminate oxidation that is mediated by O 2 • − from that mediated by NO• (Zielonka et al, 2012b).…”

“…Although O 2 • − can directly oxidize HE to its radical, the reaction is relatively slow and is more likely to be performed by other cellular oxidants present (Winterbourn, 2014 the non-specific reaction, any specificity of the fluorescent signal for O 2 • − needs to be confirmed with ROS-scavenging enzymes and NOS inhibitors (Zielonka et al, 2012b).…”

“…Possible single-cell approach for ROS and RNS detection in functional HSCs -determination of HSC redox status in situ A possible assay that could be applied to stem cell biology came from a recent study suggesting that fluorescence-based techniques using HE, boronate probes, DAF-DA, can be used for initial screening before -in a second step -specificity of these probes for certain reactive species is confirmed with NOS inhibitors and ROSdetoxifying enzymes (Zielonka et al, 2012b). It should be noted that there is always uncertainty as to whether the ROS scavengers reach the site of oxidation.…”

“…The probe diaminofluorecscein (DAF-2) and its cell-permeable diacetate (DFA-2DA) are used to monitor the formation of cellular NO• based on the generation of the resulting green fluorescent product DFA-2T (Zielonka et al, 2012b). DAF-2 does not react directly with NO•, but with N 2 O 3 , which is generated upon NO• oxidation and can be attenuated in the presence of the ascorbic acid (Ye et al, 2004).…”