Background – What is ROS
Reactive oxygen species (ROS) is a normal product of cell metabolism - an oxygen-containing bioactive molecule, including peroxide, superoxide, hydroxyl radical, singlet oxygen and α-oxygen, etc., which play an important regulatory role in cell signaling pathways and transcription, such as apoptosis, autophagy, aging, cancer, etc.
Effect of ROS concentration on cells – why detect ROS?
At low levels of reactive oxygen species, ROS is involved in intracellular signaling and regulation as "redox messengers". However, under environmental stresses (e.g., ionizing radiation, heat exposure, ultraviolet rays, hypoxia, etc.), ROS levels increase dramatically, which may cause DNA damage, inhibit gene expression, lead to protein misfolding and even affect protein synthesis, causing severe damage to cellular structures, which is known as oxidative stress.
Once ROS levels exceed the capacity of endogenous antioxidant defenses, the redox balance is disrupted, leading to structural or conformational changes in DNA, lipids, proteins, and ultimately cell death.
ROS level is an important signal of cell damage caused by normal physiological functions and environmental factors, and the detection of intracellular ROS level is of great significance for understanding the signaling pathways and potential mechanisms of action of some drugs. Therefore, the selection of appropriate probes for the detection of ROS plays an important role in disease mechanism research and drug screening.
How does ROS detection work?
The Reactive Oxygen Species Assay Kit (CAT# 50101ES01) is the most commonly used method for quantifying intracellular reactive oxygen species levels based on changes in the fluorescence intensity of the fluorescent dye DCFH-DA (2,7-Dichlorodi-hydrofluorescein diacetate).
DCFH-DA itself has no fluorescence and can freely pass through the cell membrane, and after entering the cell, it can be hydrolyzed by intracellular esterases to produce DCFH. However, DCFH cannot penetrate the cell membrane, making it easy for the probe to be labeled and aggregated within the cell. Intracellular reactive oxygen species are able to oxidize non-fluorescent DCFH to form fluorescent DCF. The intensity of DCF green fluorescence is directly proportional to the level of intracellular reactive oxygen species, and the level of intracellular reactive oxygen species can be known by detecting the fluorescence of DCF.
Under the conditions of excitation wavelength of 488 nm and emission wavelength of 525 nm, DCF fluorescence was detected by fluorescence microscope, laser confocal microscope, fluorescence spectrophotometer, fluorescence microplate reader, flow cytometer, etc., so as to determine the level of intracellular reactive oxygen species.
Literature published by customers using this product (some examples)
As of September 2022, a total of 164 articles have been published in the ROS reactive oxygen species detection kit (50101ES01), with a total impact factor of 913.72.
Effect of ROS production on macrophage polarization and tumor cell killing （PMID: 35665496; PMID: 35301299; PMCID: PMC8931093PMCID: PMC9353410.
FAQ
Q1: Which samples are suitable for ROS testing?
A1: It is generally used for the detection of mammalian cells, and is only suitable for the detection of reactive oxygen species in live cells or in vivo.
Q2: Is the ROS test kit suitable for detection in serum or plasma?
A2: Not suitable for the detection of ROS in serum or tissue homogenate. Fresh tissue prepared single-cell suspensions can be tried.
Q3: Can plants or bacteria be detected?
A3: It is only suitable for the detection of reactive oxygen species in live cells or in vivo, because the half-life of hydroxyl radicals and superoxide radicals of oxygen is very short, and it is only suitable for the detection of live cells. Plants or bacteria, which can be used for detection after preparation of protoplasts, this kit cannot detect ROS in vivo.
Q4: How can I avoid excessive fluorescence background?
A4: After probe incubation, be sure to wash away any remaining probes that have not entered the cell.
Q5: Can I detect the amount of ROS in normal cells?
A5: The content of reactive oxygen species in normal cells is very low, and the detection effect may not be very good.
Q6: The negative and positive fluorescence values are the same, what is going on?
A6: It may be caused by the concentration of the probe added is too large, it is recommended to reduce the probe concentration by 5-7.5 μM and the incubation time: 15-20 min.
Q7: The fluorescence of the positive control is weak, what is going on?
A7: The positive control Rosup was usually concentrated at 100 μM, and a significant increase in reactive oxygen species was observed 30 min-4 h after stimulation. The effect of the reactive oxygen species positive control may vary greatly between different cells. If the increase in ROS is not observed within 30 minutes after stimulation, the induction time can be extended or the concentration of Rosup can be increased appropriately.
A8: The same probe, not divided, the first 5 times the effect is very good, this time it is not dyed, what is the matter?
Q8: 1. The cell state is not good, resulting in low staining efficiency; 2. The induction time of positive drug is too short, and the level of reactive oxygen species can be significantly increased by incubating at 37°C in the dark for 30 min-4 h; 3. The probe has been frozen and thawed more than 4 times, the staining efficiency is reduced, and the fluorescence signal is unstable (sometimes strong, sometimes weak, and easy to quench). It is recommended that the probes be aliquoted and stored in a -20°C freezer protected from light to avoid repeated freeze-thaw cycles.
Q9: What instruments can be used for testing?
A9: Fluorescence microscope, laser confocal microscope, fluorescence spectrophotometer, fluorescence microplate reader, flow cytometer, etc. can detect fluorescence values.
Scientific research publications published by customers using this product (partial)
[1] Zhong D, Jin K, Wang R, Chen B, Zhang J, Ren C, Chen X, Lu J, Zhou M. Microalgae-Based Hydrogel for Inflammatory Bowel Disease and Its Associated Anxiety and Depression. Adv Mater. 2024 Jan 26: e2312275. doi: 10.1002/adma.202312275. Epub ahead of print. PMID: 38277492. IF: 29.4
[2] Zhang M, et al. Conscription of Immune Cells by Light-Activatable Silencing NK-Derived Exosome (LASNEO) for Synergetic Tumor Eradication. Adv Sci (Weinh). 2022 Aug;9(22): e2201135. doi: 10.1002/advs.202201135. Epub 2022 Jun 4. IF: 16.806
[3] Zhang D, et al. Microalgae-based oral microcarriers for gut microbiota homeostasis and intestinal protection in cancer radiotherapy. Nat Commun. 2022 Mar 17;13(1):1413. doi: 10.1038/s41467-022-28744-4. PMID: 35301299. IF: 14.919
[4] Jiao D, et al. Biocompatible reduced graphene oxide stimulated BMSCs induce acceleration of bone remodeling and orthodontic tooth movement through promotion on osteoclastogenesis and angiogenesis. Bioact Mater. 2022 Feb 6; 15:409-425. doi: 10.1016/j.bioactmat.2022.01.021. PMID: 35386350; PMCID: PMC8958387. IF: 14.593
[5] Guo G, et al. Space-Selective Chemodynamic Therapy of CuFe5O8 Nanocubes for Implant-Related Infections. ACS Nano. 2020 Oct 27;14(10):13391-13405. doi: 10.1021/acsnano.0c05255. Epub 2020 Sep 22. PMID: 32931252. IF: 14.588
[6] Yang C, et al. Red Phosphorus Decorated TiO2 Nanorod Mediated Photodynamic and Photothermal Therapy for Renal Cell Carcinoma. Small. 2021 Jul;17(30): e2101837. doi: 10.1002/smll.202101837. Epub 2021 Jun 19. PMID: 34145768. IF：13.281
[7] Xiaolu Chen, et al. Metal-phenolic networks-encapsulated cascade amplification delivery nanoparticles overcoming cancer drug resistance via combined starvation/chemodynamic/chemo therapy. Chemical Engineering Journal. 2022 Aug; 442:136221. IF: 13.273
[8] Hao Ding, et al. Mesenchymal stem cells encapsulated in a reactive oxygen species-scavenging and O2-generating injectable hydrogel for myocardial infarction treatment. Chemical Engineering Journal. 2022.133511:1385-8947. IF: 13.273
[9] Yu H, et al. Triple cascade nanocatalyst with laser-activatable O2 supply and photothermal enhancement for effective catalytic therapy against hypoxic tumor. Biomaterials. 2022 Jan; 280:121308. PMID: 34896860. IF: 12.479
[10] Sun D, et al. A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer. Acta Pharm Sin B. 2022 Jan;12(1):378-393. PMID: 35127393. IF: 11.614
[11] Xiong Y, et al. Tumor-specific activatable biopolymer nanoparticles stabilized by hydroxyethyl starch prodrug for self-amplified cooperative cancer therapy. Theranostics. 2022 Jan 1;12(2):944-962. PMID: 34976222. IF: 11.556
[12] Gao J, et al. Mitochondrion-targeted supramolecular "nano-boat" simultaneously inhibiting dual energy metabolism for tumor selective and synergistic chemo-radiotherapy. Theranostics. 2022 Jan 1;12(3):1286-1302. PMID: 35154487. IF: 11.556
[13] Zhong D, et al. Calcium phosphate engineered photosynthetic microalgae to combat hypoxic-tumor by in-situ modulating hypoxia and cascade radio-phototherapy. Theranostics. 2021 Jan 22;11(8):3580-3594. PMID: 33664849. IF: 11.556
[14] Sun J, et al. Cytotoxicity of stabilized/solidified municipal solid waste incineration fly ash. J Hazard Mater. 2022 Feb 15;424(Pt A):127369. doi: 10.1016/j.jhazmat.2021.127369. Epub 2021 Sep 29. PMID: 34879564. IF: 10.588
[15] Zhu C, et al. Multifunctional thermo-sensitive hydrogel for modulating the microenvironment in Osteoarthritis by polarizing macrophages and scavenging RONS. J Nanobiotechnology. 2022 May 7;20(1):221. IF: 10.435
[16] Pan X, et al. Zinc oxide nanosphere for hydrogen sulfide scavenging and ferroptosis of colorectal cancer. J Nanobiotechnology. 2021 Nov 27;19(1):392. doi: 10.1186/s12951-021-01069-y. PMID: 34838036; PMCID: PMC8626909. IF: 10.435
[17] He J, et al. Gold-silver nanoshells promote wound healing from drug-resistant bacteria infection and enable monitoring via surface-enhanced Raman scattering imaging. Biomaterials. 2020 Mar; 234:119763. PMID: 31978871. IF: 10.317
[18] Cheng Q, et al. Nanotherapeutics interfere with cellular redox homeostasis for highly improved photodynamic therapy. Biomaterials. 2019 Dec; 224:119500. doi: 10.1016/j.biomaterials.2019.119500. Epub 2019 Sep 17. PMID: 31557591. IF: 10.273
[19] Zhong D, et al. Laser-triggered aggregated cubic α-Fe2O3@Au nanocomposites for magnetic resonance imaging and photothermal/enhanced radiation synergistic therapy. Biomaterials. 2019 Oct; 219:119369. PMID: 31351244. IF: 10.273
[20] Sun C, et al. Selenoxide elimination manipulate the oxidative stress to improve the antitumor efficacy. Biomaterials. 2019 Dec; 225:119514. doi: 10.1016/j.biomaterials.2019.119514. Epub 2019 Sep 24. PMID: 31569018. IF: 10.273
Recommended products
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Product name |
Catalog number |
Size |
|
Reactive Oxygen Species Assay Kit |
50101ES01 |
1 Kit (1000 tests) |