Reactive oxygen species detection

June 3, 2019

Reactive oxygen species are measured in cancer research as well as in immunology or in food laboratories. This proves that ROS has many causes and just as many effects. These can be both physiological and pathological. This blog post explains what reactive oxygen species are and how they can be measured.

Dr Andrea Krumm

What are reactive oxygen species (ROS)?

Reactive oxygen species are unstable molecules, which contain oxygen as a byproduct of the natural metabolism of oxygen. These molecules are very reactive and come in many different forms, examples include: H₂O₂ (hydrogen peroxide), NO (nitric oxide), O₂^-(oxide anion), peroxynitrite (ONOO^-), hydrochlorous acid (HOCl), and hydroxyl radical (OH^-)¹.

When reactive oxygen species build up at high levels inside cells, also referred to as oxidative stress, they can damage DNA, RNA and proteins, and even cause cell death. ROS biomolecules have been implicated in a range of pathologies, including cancer, neurodegenerative diseases, atherosclerosis and the process of ageing². The body responds to oxidative stress by increasing production of antioxidants such as catalase and glutathione which convert dangerous free radicals to harmless molecules, such as water³.

Low levels of reactive oxygen species have been found to have useful and beneficial effects. For example, they play an important role in regulating cellular signaling and gene expression. Evidence suggests that reactive oxygen species may modulate cell differentiation, including hematopoietic differentiation involving stem cell production, in both physiological and pathological (disease) conditions⁴.

There are many intracellular sources of reactive oxygen species, including cell mitochondria and NADPH oxidases (NOX enzymes) linked to neutrophils², white blood cells, which produce high levels of ROS as part of their defense role. A wide range of enzymes also produce ROS, including xanthine oxidase, nitric oxide synthase, cyclooxygenases, cytochrome P450 enzymes and lipoxygenases.

Oxidative stress is further induced by exogenous stimuli. For instance, alcohol leads to formation of reactive oxygen species during its degradation and induce ROS production by activation of cytochrome P450 enzymes⁵. A second example of a drug inducing ROS is tobacco smoke. It contains radicals reacting with oxygen to form ROS⁶. An exogenous ROS-inducer that is less avoidable is ultraviolet light. The induction of reactive oxygen species increases oxidative damage to cellular components and thus contributes to the development of skin cancer⁷.

Assays detecting reactive oxygen species

As reactive oxygen species impact on many biological events, they are studied extensively. Assays to measure ROS belong to the category of cell-based assays and are used to report on immune responses, to monitor the balance of ROS and antioxidants or to study their role in cancer development. However, detecting ROS levels can be difficult as they are often in the nanomolar range and have very short half-lives.

The most common reagent used for measuring ROS is 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). It is intracellularly trapped by esterase activity that removes lipophilic blocking groups. Upon oxidation of the probe by ROS the resulting dichlorofluorescein (DCF) exhibits high fluorescence. Different derivatives of H₂DCFDA are employed for reactive oxygen species measurements as they have specific characteristics: carboxy-H₂DCFDA displays an improved cellular retention whereas the fluorinated molecule (H₂DFFDA) proves more stable when exposed to light.

A probe that selectively detects superoxide generated in mitochondria during oxidative phosphorylation is MitoSOX™ Red (ThermoFisher). The live cell reagent is targeted to mitochondria and its oxidation by superoxide increases its red fluorescence.

Apart from fluorescent probes added to the cell culture, genetically encoded probes may report on changes in reactive oxygen species directly in the cell at the location of interest. The fluorescent probe roGFP (redox sensitive green fluorescent protein) has different excitation wavelengths dependent on its redox state: reduced roGFP can be excited at 488 nm whereas oxidized roGFP is better excited at 405 nm (Fig. 2). A peroxidase that scavenges selectively H₂O₂ facilitates oxidation of roGFP and the ratio of measurements performed with both excitation wavelengths reports on the presence of H₂O₂⁸. A similar approach is employed by the genetically encoded H₂O₂ sensor HyPer. The Biosensor thiazole orange in the antioxidant assay kit AOP-1 also functions as a biosensor to detect ROS. At the same time it also fills the role of a photosensitizer, generating ROS in living cells, which is a prerequisite to study the presence and effects of potential antioxidants.

In the scientific talk "Real-time monitoring of redox changes in cells with a microplate reader", Bruce Morgan, Professor for Biochemistry at the University of Saarbrücken, Germany, discusses how redox-sensitive probes can be used to monitor redox enzyme activity. To address this question, roGFP2 was combined with glutaredoxin or glutathione. His team was then able to induce the oxidation of the signal molecule and to monitor in semi-high-throughput the change of roGFP fluorescence on a CLARIOstar Plus microplate reader.

Next to fluorescent methods, luminescent methods are available to detect reactive oxygen species. On the one hand, there are methods based on chemiluminescence. They employ a molecule such as luminol, lucigenin, Pholasin^® or the recently described and more sensitive L-012 that emit light when transitioning to their oxidized state. As this process is induced by ROS, light emission can be directly translated to the presence of ROS⁹. On the other hand, enzyme-dependent luminescence (bioluminescence) detects ROS. The enzyme is a luciferase that converts a substrate under the emission of light. A precursor of the substrate is added to the sample and is ROS-dependently converted into the luciferase substrate. Thus the luminescent signal correlates with ROS levels¹⁰. Another example is highlighted in this application note, that employed Promega’s luminescent ROS-Glo assay for the detection of ROS in cell-based assays.

Instrumentation required for detecting reactive oxygen species

These popular methods to study and measure levels of reactive oxygen species are based on fluorescent or luminescent read-outs. This makes it possible to use a microplate format allowing running 96 up to 1536 samples at once and performing the detection in microplate readers. These devices record light signals in the tiny reactions taking place in the wells of a microplate.

Dectection modes: depending on the assay chosen to detect reactive oxygen species the ideal microplate reader needs to cover either luminescence, fluorescence or both if the flexibility running different assays should be given. However, if you would like to combine the Reactive oxygen species measurement with other assays, further detection modes may need to be covered as well. An example would be the combination of ROS-measurements with absorbance-based viability assays such as WST. BMG LABTECH offers a range of multi-mode plate reader capable of measuring luminescence, fluorescence, absorbance and advanced detection modes.

Sensitivity: The sensitivity the ideal microplate reader needs to offer is dictated by the plate format that is used and by the ROS-assay itself. For instance high density plate formats (1536 well) have a lower reaction volume and lower signal intensities. This requires highest sensitivity of the microplate reader and a fast reading of the plate. The PHERAstar FSX microplate reader is designed to meet exactly these requirements.

The most commonly used Reactive oxygen species assay, the H2DCFDA assay, gives high signal intensities when used in 96-well or 384-well format and with sufficient material, for instance when using immortal cell lines. For instance, the signals can be easily recorded by all BMG LABTECH multi-mode readers. Increased sensitivity is required by genetically encoded probes such as roGFP since expression levels are limited. Researcher Prince Saforo Amponsah from the Technical University Kaiserslautern explains in detail the challenge to read the fluorescent probe and how the CLARIOstar^® assisted the measurements.

Accessories: Reactive oxygen species detection based on H₂DCFDA, bioluminescence or roGFP is suitable to be kinetically measured in live cells. Depending on the time frame of interest, live cell measurements require a gas atmosphere to allow long-term cell incubation. In particular this is a CO₂ atmosphere of 5 10 % to guarantee a stable pH in carbonate buffered media. For the measurement of reactive oxygen species the surrounding O₂ concentration plays a critical role. The atmospheric O₂ level of 21 % alone is capable of inducing oxidative stress without further stimulation. Physiologic O₂ concentrations that cells are exposed to in the human body are much lower ranging from 4 % in the brain and 10 % in the kidney¹¹. Accordingly, reliable ROS measurements can only be achieved when measuring at lower O₂ concentrations. The atmospheric control unit (ACU) regulates both the CO₂ and O₂ concentration in the microplate reader and thus allows running ROS experiments at physiological O₂. The tool is available for CLARIOstar, VANTAstar and Omega microplate readers.

If you are interested in real-life data measuring reactive oxygen species on BMG LABTECH readers, please feel free to have a look into our collection of peer-reviewed articles on this topic.