Reactive oxygen species (ROS) are oxygen-containing byproducts generated during cellular metabolism. Under physiological conditions, ROS levels are tightly regulated and play roles as signaling molecules in normal cell signal transduction, cell cycle progression, gene expression, and maintaining homeostasis. However, high concentrations of ROS induce oxidative stress, leading to damage of cellular macromolecules such as DNA, membrane lipids, and proteins. This oxidative damage can trigger apoptosis and is also associated with aging, cancer, neurodegenerative diseases, diabetes, and inflammation.
Q1: What are reactive oxygen species (ROS)? What types exist?
ROS refers to oxygen-containing free radicals and peroxides that are related to oxygen metabolism in organisms, including peroxides, superoxides, hydroxyl radicals, singlet oxygen, and α-oxygen species.
Examples include: superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (·OH), hypochlorous acid (HOCl), peroxynitrite anion (ONOO⁻), and nitric oxide (NO).
Q2: What are the detection methods for ROS?
ROS detection methods can be divided into direct and indirect approaches:
① Direct detection generally uses specific fluorescent probes;
② Indirect detection involves measuring damage to lipids, proteins, or DNA;
③ Alternatively, detecting antioxidant levels and related enzyme activities can be used.
Q3: What are the common probes used for direct ROS detection?
There are five major types:
- Total ROS – DCFH-DA (H₂DCFDA), green fluorescence
- Total ROS – CellROX Orange, orange fluorescence
- Superoxide anion – DHE, red fluorescence
- Mitochondrial ROS – MitoSOX, red fluorescence
- Nitric oxide (NO) – DAF-2 DA, green fluorescence
Q4: Can DCFH-DA be used for bacteria, fungi, or plants?
DCFH-DA is different from antibodies—it is not species-specific. Moreover, due to its small molecular weight, its use in E. coli for ROS detection has been validated in the literature.
Q5: What should I do if there’s no positive signal when detecting with DCFH-DA?
If no significant positive signal or positive cell population is observed when using DCFH-DA to detect ROS compared to the control group, consider the following troubleshooting points:
Check for the presence and outcome of a positive control.
Common positive controls for total ROS include hydrogen peroxide (H₂O₂), tert-butyl hydroperoxide (TBHP), antimycin A (AMA), and pyocyanin.
For example, in Figure A, pyocyanin was used as the positive control for Huh7 cells. Both ROS (DCF) and superoxide (DHE) signals were increased [1].
Check the working concentration and incubation conditions of DCFH-DA.
Low working concentrations of DCFH-DA can lead to weak signals. You may increase the concentration from 1 μM to 2 μM or 4 μM. A typical incubation condition is 37°C for 30 minutes.
[Note]: Dilution buffer can be PBS, HBSS, or serum-free medium.
Sample handling for cell-based assays.
Whether using DCFH-DA, DHE, or MitoSOX Red, these probes are intended for live cell detection.
Do not fix samples, and do not use anti-fade mounting media or perform coverslipping after staining.
DCFH-DA staining principle: Non-fluorescent DCFH-DA freely permeates the cell membrane and is hydrolyzed by intracellular esterases to form non-fluorescent DCFH. In the presence of ROS, DCFH is oxidized to fluorescent DCF, and green fluorescence intensity correlates with ROS levels.
Q6: How do I choose a suitable positive control?
Positive controls vary depending on the type of ROS being studied. For MitoSOX, the recommended positive control is MitoPQ, and the negative control is DETA NONOate.
Table 1: Positive and Negative Controls for ROS Detection
|
ROS Type |
Positive Control |
Negative Control |
|
Total ROS |
TBHP (tert-butyl hydroperoxide), H₂O₂ |
– |
|
ROS; Superoxide |
AMA (Antimycin A) |
– |
|
Superoxide; Hydrogen Peroxide |
Pyocyanin |
– |
|
Nitric Oxide |
LPS (lipopolysaccharide) |
– |
|
Mitochondrial Superoxide |
MitoPQ |
DETA NONOate |
Q7: Which nuclear dye should be used if I need to stain the nucleus?
For live cell imaging, Hoechst 33342 is recommended due to its membrane permeability. Although DAPI is also a nuclear dye, Hoechst is preferred for live cell applications. Both emit blue fluorescence.
Q8: How should the detection results be analyzed?
Results can be analyzed by comparing the average fluorescence intensity of ROS across different experimental groups. Additionally, ROS-positive cell percentages can be calculated for comparative analysis.
Reference
Lynnyk A, Lunova M, Jirsa M, Egorova D, Kulikov A, Kubinová Š, Lunov O, Dejneka A. Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation. Biomed Opt Express. 2018 Feb 21;9(3):1283–1300. doi:10.1364/BOE.9.001283. PMID: 29541521; PMCID: PMC5846531.
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