Biochemistry

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), induce oxidative stress. This stress plays crucial roles in cellular signaling, stress response, and disease progression, making the quantification of free radicals essential for understanding oxidative stress mechanisms. Here, we present a high-throughput fluorescence-based protocol for measuring the presence of total free radicals, including ROS and RNS, in the whole adult Drosophila melanogaster (fruit fly). The protocol involves homogenizing whole adult flies in PBS and treating only the supernatant of the lysate with dichlorodihydrofluorescein-DiOxyQ (DCFH-DiOxyQ), which then converts into a fluorescent molecule, dichlorofluorescein (DCF), upon reacting with free radicals. The level of fluorescence is directly proportional to the amount of free radicals present in the sample. This protocol offers simplicity, scalability, and adaptability, making it ideal for studying oxidative stress in the model organism Drosophila and its different tissues under different dietary regimes, environmental stresses, genetic mutations, or pharmacological treatments. It is to be noted that the protocol uses a kit from Abcam, which has been used to measure free radicals in mice, rats, human blood, and cell lines. It can also be applied to biofluids, culture supernatants, and cell lysates, making it suitable for a wide range of sample types beyond whole organisms or tissues. However, due to our research focus and expertise, here we describe a detailed protocol to measure free radicals responsible for inducing oxidative stress only in fruit flies.

Macrophages are at the center of innate immunity and are the main target cells of the intracellular pathogen Salmonella enterica serovar Typhi. The production of reactive oxygen and nitrogen species (ROS/RNS) is the host’s early response to invading microbes, as oxidative stress is highly toxic for bacteria. Adequate ROS/RNS production in infected macrophages is critical for the clearance of intracellular pathogens; this is achieved by several enzymes, including inducible NADPH phagocyte oxidase (NOX) and nitric oxide synthase (iNOS), respectively. The pro-inflammatory cytokine interferon gamma (IFNγ), primarily produced by activated natural killer cells and T-helper cells type 1, is a potent inducer of iNOS. Therefore, it is crucial for infection control through oxidative microbicidal activity.

To characterize the early oxidative stress response via ROS formation, which is critical for the reduction of Salmonella proliferation within macrophages, we established an in vitro model of murine macrophages infected with Salmonella enterica serovar Typhimurium (S.tm). This serovar induces a systemic infection in mice that is frequently used as a model for typhoid fever, which, in human subjects, is caused by Salmonella Typhi.

We generated bone marrow–derived macrophages (BMDM) from C57BL/6N wildtype mice using macrophage colony-stimulating factor (M-CSF) stimulation for six days. Thereafter, we infected BMDM with S.tm for one hour. Shortly before infection, cells were stained with CellROX^TM Deep Red reagent. In its reduced form, CellROX^TM is non-fluorescent. As a result of oxidation by ROS, this reagent exhibits strong fluorescence and persists within the cells. Subsequently, changes as a result of the oxidative stress response can be measured with a TECAN Spark microplate reader over time.

We designed this protocol to measure oxidative stress in macrophages through the course of an infection with an intracellular bacterium. The protocol has several advantages over established techniques. First, it allows to continuously monitor and quantify ROS production in living cells from the very start of the infection to the final clearance of the intracellular pathogen. Second, this protocol enables efficient ROS detection without stressing the cells by detaching or staining procedures.

Graphical abstract

Depending on its local concentration, hydrogen peroxide (H₂O₂) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H₂O₂ are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of H₂O₂ in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular H₂O₂ dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent H₂O₂ sensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local H₂O₂ dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader.

Graphical abstract:

Overview of HyPer7 sensor function and measurement results.

Reactive oxygen species are ubiquitous in nature, and function as signalling molecules in biological systems; they may also contribute to oxidative stress in several pathobiological disease states. In this report, we describe a simple, reliable, sensitive, and specific assay for the detection and quantitation of hydrogen peroxide (H₂O₂) release by living cells, organoids, or tissues. Furthermore, the low cost of reagents required for this assay makes it inexpensive relative to commercial kits. The high sensitivity and specificity are based on the ability of H₂O₂ to react with heme peroxidases and convert para-substituted phenolic compounds to fluorescent dimers.

Graphical abstract:

Reagents such as Amplex^® Red have been developed for detecting hydrogen peroxide (H₂O₂) and are used to measure the release of H₂O₂ from biological samples such as mammalian leukocytes undergoing the oxidative burst. Caenorhabditis elegans is commonly used as a model host in the study of interactions with microbial pathogens and releases reactive oxygen species (ROS) as a component of its defense response. We adapted the Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit to measure H₂O₂ output from live Caenorhabditis elegans exposed to microbial pathogens. The assay differs from other forms of ROS detection in the worm, like dihydrofluorescein dyes and genetically encoded probes such as HyPer, in that it generally detects released, extracellular ROS rather than intracellular ROS, though the distinction between the two is blurred by the fact that certain species of ROS, including H₂O₂, can cross membranes. The protocol involves feeding C. elegans on a lawn of the pathogen of interest for a period of time. The animals are then rinsed off the plates in buffer and washed to remove any microbes on their cuticle. Finally, the animals in buffer are distributed into 96-well plates and Amplex^® Red and horseradish peroxidase (HRP) are added. Any H₂O₂ released into the buffer by the worms will react with the Amplex^® Red reagent in a 1:1 ratio in the presence of HRP to produce the red fluorescent excitation product resorufin that can be measured fluorometrically or spectrophotometrically, and the amount of H₂O₂ released can be calculated by comparison to a standard curve. The assay is most appropriate for studies focused on released ROS, and its advantages include ease of use, the ability to use small numbers of animals in a plate reader assay in which measurements can be taken either fluorometrically or spectrophotometrically.

The intestine is endowed with an innate immune system that is required to fight any exogenous bacteria that are swallowed along with the food. The first line of defense that is mounted by the gut epithelium is the release of immune Reactive Oxygen Species (ROS), such as hypochlorous acid (HOCl), into the lumen. HOCl is produced within 1.5 h of bacterial ingestion and is very labile once released. Therefore, to monitor HOCl production upon ingestion of allochthonous bacteria, one needs a detection system that can quickly and efficiently detect HOCl production in the intestine. While most of the ROS-sensitive probes available in the market detect all kinds of ROS without any distinction, the R19-S fluorescent probe has been developed to specifically detect HOCl. Here, we describe a protocol to monitor HOCl production using this probe in the gut lumen of adult Drosophila upon ingestion of the opportunistic bacteria Bacillus thuringiensis.

Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ROS by using an oxidant-sensing probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in cyanobacteria. This method provides reliable, simple, rapid and cost effective means for detection of ROS in cyanobacteria.

Oxidative stress has been proposed to be one of the main causes of aging and has been implicated in the pathogenesis of many diseases. Sensitivity to oxidative stress can be measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as paraquat or juglone. Sensitivity to oxidative stress is a balance between basal levels of ROS, the ability to detoxify ROS, and the ability to repair ROS-mediated damage.

Superoxide ions (O^2-) and hydrogen peroxide (H₂O₂) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O^2- ions is determined qualitatively using nitrobluetetrazolium (NBT) assay while the H₂O₂ is qualitatively estimated using 3,3-diaminobenzidine (DAB) and 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) assay. Further the aqueous content of H₂O₂ is estimated quantitatively using ferrous oxidation-xylenol orange (FOX) assay.

This protocol describes the measurement of hydrogen peroxide (H₂O₂) content in Arabidopsis root tissue by using the Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit. When root tissue is disrupted and resuspended in phosphate buffer, H₂O₂ is released from the cells. The obtained root extracts containing H₂O₂ can be mixed with a solution containing Amplex^® Red reagent (10-acetyl-3,7-dihydrophenoxazine). In the presence of horseradish peroxidase, the Amplex^® Red reagent reacts with H₂O₂ in a 1:1 stoichiometry. The resulting product is the red-fluorescent compound resorufin which can be detected fluorometrically or spectrophotometrically. Our protocol is based on the manual of the Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit and describes a step-by-step procedure with a detailed description of the necessary controls and data analysis. We have also included modifications of the protocol, notes and examples that intend to aid the user in easily reproducing the assay with their own samples.