Science topic

Reactive Oxygen Species - Science topic

Molecules or ions formed by the incomplete one-electron reduction of oxygen. These reactive oxygen intermediates include SINGLET OXYGEN; SUPEROXIDES; PEROXIDES; HYDROXYL RADICAL; and HYPOCHLOROUS ACID. They contribute to the microbicidal activity of PHAGOCYTES, regulation of signal transduction and gene expression, and the oxidative damage to NUCLEIC ACIDS; PROTEINS; and LIPIDS.
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There are many proxies to measure the firm performance of private sector firms, such as ROA, ROE, ROI, ROCE, ROS, etc. How to measure the performance of public sector firms, please? Could anyone suggest something, please? Suggestions, recommendations, and comments are welcome. Thank you
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Dear Hans-Georg Petersen, Thank you very much for the suggested material.
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How to calculate fish ROS activity from absorbance? The ROS level was measured by the modified dichlorofluorescein-diacetate (DCF-DA) method. Can anyone guide me with the calculation through excel sheet?
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Hello, I am vet, I performed the experiment on animals and results.
Thank you,
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i am coonducting research on wheat to determine salt stress on triticum aestivum while tea residue as mistication agent .want to know how plant respond to stress genetically?
How reactive oxygen species are produced?
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Salinity imposes stress by damaging ionic and osmotic balances in plants. Osmotic stress caused by increasing the amount of salt in soil, decreases the amount of water that plant use and as a result physiological drought occurs.
More details can be accessed at:
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Dear researcher
I have obtained my OD at 485 and 522 nm. I don't understand how to interpret the data. Kindly help me to calculate ROS
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thank you Juan Manuel Gallardo sir !
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We want to analyze whether T cells produce ROS under certain pahtophysiologic conditions.
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Try this for method and alternatives
best of luck
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I am looking for information on ROS detection methods that is not a secondary protein change or etc from the ROS presence. Would like to use it in live tissue and it remain stable after PFA and detergents treatment. Thanks in advance.
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Hi Alexandria E Ostman,
This is extremely difficult because ROS have only extremely short-life time. Direct detection can be done "in vivo" with probes such as ESR or NMR (MRI), or in sections or cells with ROS-reactive fluorescent probes. The latter has considerable artifacts and quantitative problems. However, several products such as DCFDA and mitoSOX are available from Molecular Probes. Note that reactivity with individual ROS and resistance to fixing processes differ for each probe.
For blood cells these probes can be detected quite quantitatively by flow cytometry.
Because of these problems, it would be better to include additional evaluations to increase reliability such as simultaneous evaluation of adducts and other denatured products of proteins resulting from reactions caused by ROS at the same time.
Good luck!
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Facing an issue with H2o2 control experiments with cancer cell line. Could anyone help me.
Thanks
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@Philip and jochem spieker Thank you for your answers. I will give a try.
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please provide some papers or standardized protocols to check ROS activity in blood samples.
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Dear Navdeep Singh, please see the enclosed files.
Best regrds,
I. Popov
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Before neurotoxicity with ethanol, giving an excess of antioxidants (vitamin E, DHA, carotenoids) previous days before intoxication can hinder the ability of the enzymes (because the antioxidants are already dealing with ROS and RNS) to deal with sudden the oxidative stress?
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Hello, I need to measure ROS and NO in plasma samples.
I have a fluorescence reader for ROS and reagents for the Griess test for NO.
Do you have a protocol for the preparation of the samples?
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Here an example
I hope it works for you
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I couldn't see much paper where plant breeders use biochemical such as proline content Malondialdehyde (MDA) and dyes such as NBT or DAB( for ROS detection) for screening stress-tolerant accession on a large scale (100-200 which I suppose is possible to do). Are not these methods better than phenotyping grain yield, biomass, plant height, NDVI, LAI, etc?
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The biochemical analysis are important, but if you have plant breeding programme should be more interesting and effective to make evaluation on the field. In those conditions you can evaluate and select the best plants for abiotic stress plants, and maybe to others topics like vigorous plants, also for production and quality. Then, you can select the most promissed material for your proposed objectivesz.
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There is a antioxidant defense system in cell.
Have any signalling pathway or other things can influence ROS level in a extracelluar way?
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Hi, the pathways of PAMPs and DAMPs mediated by pattern recognition receptors such as TLRs are missing. These can be directly oxidative stress in the intracellular pathway through the classical NFκB pathway or inflammasome-mediated inflammation, or they can induce inflammation through producing proinflammatory cytokines.Hypoxic response (mainly Hif-1α) and oxidative stress from mitochondria during ischemia-reperfusion should also be considered.
Thanks! 
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I was wondering if anyone has documented the generation of reactive oxygen species (ROS) in growth media under light irradiation, with the ROS generated by endogenous photosensitisers in growth media?
I'm curious about what photosensitisers are being utilised here, and what I can do to prevent this in in-vitro photodynamic therapy experiments. Ideally we would like no ROS under light irradiation alone.
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No, I can not tell you some
about it.
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For determination of ROS in mitochondrial fraction using DCFDA, How to quantify the concentration of isolated mitochondrial fraction? using nanospectrometer or some other technique ?
If i perform BCA then it will give protein concentration in mitochondria....
But in that protein fraction, i don't think ROS production would b getting accessed.
So, if i hv isolated mitochondrial fraction, then how can i quantify its concentration?
As for DCFDA, i need some specific concentration of mitochondrial fraction (per se: 1mg/ml), so how to determine that concentration?
as in protocol they hv not mentioned anywhere that i need to hv mitochondrial protein fraction (1mg/ml)...they just said: mitochondrial fraction...
so without BCA how can i quantify my isolated mitochondrial fraction easily and quickly so that i can proceed my mitochondrial fraction for further experiments like ROS or membrane potential etc...
kindly confirm
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Hello Asha,
Let me clear you that the DCFH-DA works in the presence of intracellular esterase, hence the intact live cells are used to analyze intracellular H2O2 and other ROS. Indeed, DCFH-DA is commonly used for detecting intracellular H2O2.
DCFH-DA is a cell-permeable ester and is hydrolyzed inside the cell to the dihydroxy-DCFH, which is retained. Despite the popularity of this assay, it cannot be reliably used to measure intracellular H2O2 and other ROS for the following reasons: (i) DCFH does not directly react with H2O2 (ii) several one-electron oxidizing species will oxidize DCFH to DCF (iii) DCF can actually produce O2•− and H2O2 via reaction of DCF radical with the oxygen, thus artificially elevating the very ROS that it is attempting to quantify; (iv) transition metals, cytochrome c, and heme peroxidases can catalyze DCFH oxidation. Hence, due to these reasons, DCHF-DA should not be used as a reliable measure of H2O2.
The isolation of mitochondrial fraction is not easy and may have contamination with ER etc. You can have semi-pure mitochondria fraction but it is possible once you rupture cells of your interest and collect the semipure fraction using gradient centrifugation. And ROS is highly reactive, hence it is recommended that measurement (quantification) must be done in cell lysates as early as possible in order to avoid any changes in ROS.
The new method has been developed by Kalyanaraman and colleagues in 2003. Following this original report, DHE has been modified to allow detection of O2•− in the mitochondria by addition of a triphenylphosphonium group, which promotes its accumulation in the mitochondria. This modified DHE analog is referred to as mitoSOX, and has become commonly used for detection of O2•− within the mitochondria. The mitochondrial H2O2 can also be detected by fluorescent protein-based redox probes. There are several other methids by which the mitochondrial ROS level can be analyzed.
To quantify the mitochondrial protein content, normally BCA assay is used but I wonder if the mitochondrial protein content level give you any clue for ROS level and that too in isolated mitochondria fraction. Hence, I would suggest you to analyze the total ROS level using DCHF-DA dye in live cells under fluorescence microscope.
Best
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Let's say we have a hypoxic event in the brain which lasts under 0.5 seconds. Is this duration sufficient for ROS formation?
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Answer in simple words - The rate of chemical reaction (organic or inorganic) due to a stressor, even for 0.5 seconds, is more than enough for the formation of ROS. But if you try to quantify ROS, then you really need some next level infrastructure for evaluating the extent of ROS formation.
Why so ? Firstly, because this question is obviously related to an in vivo model and secondly by the time you complete exposure period of 0.5 seconds and start the evaluation process, the recovery mode to normoxia would have already happened. This is the reason that exposure periods are recommended for a greater duration than just few seconds.
Disclaimer - There is nothing called as Zero stress environment for any factor (including hypoxia). ROS formation is a continuous process and will happen even it is for 0.00000000000001 second.
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Hello,
I would like to detect ROS using DHE in tumor sample in situ. I have thought of the following scenario:
- to inject 20 mg/kg DHE to mice ip and after 1 hour to sacrifice the mice, take out the tumor, snap freeze, cut with cryostat and visualize under the microscope.
Do you think it is appropriate for detecting ROS within the tissue?
Any comment, suggestion is appreciated!
Thank you!
Eva
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Thank you for the suggestions!
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Hello everyone,
I am trying to measure reactive oxygen species (ROS) levels of PBS-homogenated brain tissues by DCF assay. I started with control experiments by only using PBS and hydrogen peroxide (H2O2). However, I can not get reliable and consistent values on fluorescence measurement. I tried with 1000, 100, and 10 uM H2O2 with PBS (PBS + 1000 uM H2O2 + DCF) but my values are not consistent between duplicates, for example, 10 uM H2O2 levels are higher than 100 uM H2O2 levels. Besides my controls - they only include PBS and DCF - are higher than 10 uM H2O2. What would be the reason for it? Sometimes I got signals from empty wells as well, does it indicate a problem regarding fluorescence signaling? I use 96-clear well plates and wrap them in aluminum foil to protect them from light as much as possible. I also leave plates at 37 degrees for 30 minutes before reading. Thanks a lot for any suggestions and/or comments in advance.
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Thank you! Mahmut Mijit
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How to diminish the negative effects of ROS in plant cell?
Any practical idea is most welcome.
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Thank you for providing the link for the useful article.
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I am trying to measure the reactive oxygen species for a Verticillium dahlia mutant using DCFH-DA, can anybody suggests me some protocols how to do this?
thanks in advance
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Hello, I'm looking for the same protocol to use in another filamentous fungi, Fusarium. Did you find someone which worked for you?
Thanks!!
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The production of ROS (Reactive Oxygen Species) is toxic to plant cells that deviate or interfere with the normal biochemical pathways. I had targeted quantifying different forms of ROS within specified tissue such as roots and leaves.
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I want to use tissue homogenate supernatant (Not tissue) for detecting total ROS by using H2DCF-DA dye. I know it is a cell-based technique and DCFH is a fluorogenic dye that measures reactive oxygen species (ROS) activity within the cell. But would it be possible to obtain the same results from supernatant as all cellular proteins are known to be present in the supernatant?
I am willing to know has anybody tried it before and the procedure they followed.
P.S. Tissue homogenate was centrifuged at 10,000 x g for 15 min. at 4°C. Afterward, the supernatant was stored at -80°C.
Thanks
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The H2DCF-DA is mostly used to detect total ROS in live cells. The DCFDA assay protocol is based on the diffusion of H2DCFDA into the cell. It is then deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF). DCF is highly fluorescent and is detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.
There are three ways to detect total ROS in i) cell suspention in microplate using microplate Reader, ii) Cell suspension in tubes using flow cytometry, iii) in adherent cells (fluorescent microscopy).
The principle of measuring total ROS using H2DCF-DA probably does not allow to measure in supernatant of lysed cells.
In addition, ROS are short lived and measuring ROS in frozen cell lysate using H2DCF-DA may not be possible.
Best
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Hi, I'm searching for a fluorescent mitochondria marker (to be used in live confocal microscopy) that is not dependant of membrane potential nor ROS production. I'll have MitoSox to quantify ROS production but I also want to quatify mitochondria because my treatment could affect ROS production or mitochondria viability. I've read that MitoTracker Green is independant of membrane potential, but the signal can be affect by ROS. I've also found Nonyl Acridine Orange (NAO), which is not affected by ROS, but it doesn't seem clear if it's independant of the potential.
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Nonyle Acridine orange
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I am using the Abcam ROS assay kit which uses DCFDA and gives reading in fluorescence values measured on a fluorescence plate reader. Does anyone have experience with how to analyze data and plot the values to detect ROS? I am using astrocytes but regardless of that I am concerned with the data analysis part. Thanks a lot in advance, Praveen
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Hi Gisela,
I plated ~30000 cells per well. Thanks for the reply.
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Give your suggestions with publication.
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Dear Pramod Kumar Yadav many thanks for sharing this very interesting technical question with the RG community. Please note that the first article suggested by Shin Murakami is freely available as public full text on RG:
Germ cell depletion from mammalian ovary: Possible involvement of apoptosis and autophagy
Also please have a look at the following potentially useful article:
Fate of the germ cells in mammalian ovary: A review
This review article has also been posted by the authors as public full text on RG, so that you can freely download it as pdf file.
Good luck with your work and please stay safe and healthy!
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I'm trying to mesure total ROS production in bone marrow derived dendritic cells with H2DCF-DA (Invitrogen), but I having some problems.
After differenciated, I plate the cells to 1x10^6/ml and treat them for 18hs with LPS 100ng/ml or left untreated. Next, I stain the cells with the surface marker CD11c-APC and a Live/Dead dye, wash them with PBS and finally incubate with 10uM H2DCF-DA in PBS for 30 minutes at 37°C, protected from light. I wash the cells with cold PBS, resuspend them in PBS-FBS2% and aquire them inmediatelly in a BD FacsCantoII.
The problem is that my untreated cells show a much higher geometric mean that the LPS treated cells.
I've tried different protocols, including resuspending the cells in RPMI-FBS10% before adding the H2DCF-DA in PBS, but I didn't have any luck.
I would really appreciate any help or suggestions.
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If the cells are healthy ( the cell membrane intact) PI won't get in, and the tagging mode doesn't depend on the accumulation within the cell, but the intercalation in the DNA strands. This is like double check on the cell viability.
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How can we know the ROS production when we treat bacterial cells with an antibiotic agent?
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The generation of ROS was determined using fluorogenic dye 20, 70-
dichlorofluorescein diacetate (DCFDA). About 1 mL of culture suspensions from pathogens with
cell load of 1x108 CFU mL-1
was taken into various test tubes and 2.5 μL of DCFDA solution was added. All the reaction tubes were incubated in shaker under dark condition for
25 min at 37°C. Prepared antimicrobial substance (eg.Au NPs) was added to each test tube and again 2 h incubated in dark condition. The ROS formed in the sample after 2 h was
detected at 488/525 nm of fluorescence excitation/emission intensity measured using
Fluorescence Multi-Detection Reader.
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Hi,
Just a little question about ROS. I measured ROs in untreated and treated (2T) with Ultrasound,
I've observed 2 peaks in DCFH-DA in WT. It's normal? In some experiments, the second peak appears always but variable in intensity. Do normal lymphocytes have a different basal ROS corresponding to different lymphocytes subsets?
the second question in my case, how can I interpreter my data? 2T treatments produce or not rise of ROS? should I normalize my gMFI to cell numbers?
All works report only one peak of ROS, that's for me it's strange
Ps the different count is due to loss of some lymphocyte by treatments.
thanx for suggestions
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Dear all!
What is the better way to impose cold /freezing stress to wheat plants. I need see the response of various antioxidants and ROS in wheat after exposing it to freezing/chilling temperature.
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See also the following good RG link:
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When I carried out the active species capture experiments to study the photocatalytic mechanism, I found that the amount of capture agents was different in different work, for example, in a paper, EDTA 1 mmol/L, tert-butyl alcohol (BuOH) 5 mmol/L and p-benzoquinone (BZQ) 1 mmol/L, but in another paper, their amounts are all 1mmol/l. This really confuses me, I don't know how to select the amount of capture agents in my experiments, especially for BuOH.
What do you think of this problem? Is there a general test standard or reference?
Thank you very much for your suggestions and answers.
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By the introduction of different scavenging agents into the photocatalytic system.
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Hi,
Just a little question about ROS. I measured ROs in untreated and treated (2T) with Ultrasound,
I've observed 2 peaks in DCF-DA in WT. It's normal? In some experiments, the second peak appears always but variable in intensity. Do normal lymphocytes have a different basal ROS corresponding to different lymphocytes subsets?
the second question in my case, how can I interpreter my data? 2T treatments produce or not rise of ROS? should I normalize my gMFI to cell numbers?
All works report only one peak of ROS, that's for me it's strange
Ps the different count is due to loss of some lymphocyte by treatments.
An experienced immunologist maybe could me help.
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The above paper may give you a hint. You could explore PubMed or another literature search to find out what you want.
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This dye itself has color of PE-Cy5 and after interaction with the lipid ROS, it emits green fluorescence. I have also found that this dye also interferes with the PE, PE-Cy7 channels somewhat. So I was wondering if there is an alternative to this dye? (Also, why is this the standard dye for ferroptosis related lipid peroxidation?)
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HNE
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Can activating neutrophils with IL-8 at different concentrations show different responses?
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Shin Murakami Thank you very much for your studies, professor! I used the references you sent me and I understand a lot.
📷
📷
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I want to detect ROS production in cells but spectrophotometrically, using an UV-visible spectrophotometer. As where I work we don't have a fluorescence reader (as used with the h2DCFDA probe), I wonder if you could recommend which non-fluorescent dyes or reagents can be used for this purpose.
Thanks in advance!
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I'm using abcams kit DCFDA/H2DCFDA CELLULAR ROS ASSAY KIT to measure ROS in human peripheral blood mononuclear cells by flow cytometry, but I'm having some trouble with positive control (TBHP), I have used different concentrations (50, 100 y 250uM) and negative control.
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Myeloid cells are the primary source of ROS. Neutrophils and to a lesser extent monocytes express ROS. Typical ways to activate myeloid cells includes 30 ng/ml PMA often with 2 uM ionomycin or with 1 ug/ml LPS.
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I would like to use amplex red to quantify ROS from coral tissue samples that have been snap-frozen. Is amplex red the best way to measure ROS. I will be measuring ROS from tissue samples rather than from Symbiodinium cultures. TIA
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Dear Sophia,
you are very welcome. This sounds like a good experiment to start with. Also isolated coral tissue cells of your choice might be an option.
Please feel free to ask anytime and keep me up to date :)
All the best and stay healthy,
Marc
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We are standardized the ROS detection in PBMC culture using DCFDA assay. For establish cells controls conditions, we are cultured humans PBMC during 24 hours, and 4 hours before to end the cells culture, we treat some cells with 100µM of TBHP in 10% FBS during 4 hours. After that, we takes cells with or without TBHP, treat with 2.5µM of DCFDA during 30 min, and analyzed in Flow Cytometry. Ours results are confused, because the DCFDA signal between positive and negative control are similar. Thinking, in the problem would be the concentration of TBHP; we increased the concentration to 250µM and the DCFDA signal in the positive control were less than negative control.
I would be grateful for any suggestion.
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I only used TBHP as a control in my ROS assay with DCFDA in a place reader assay. I would say it does not work every time and I don't know why. With flow cytometry, I used antimycin A as a positive control: 1 h treatment at 50µg/ml.
Good luck with your study!
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Well, we read a lot about how in vitro manipulation of oocytes can induce stress and therefore reduce their potential or as people call it "quality". I've been looking into the studies mostly, they focus on the oxidative stress and reactive oxygen species relation with calcium regulation. Other studies investigated the heating as a stress factor. Well, how they established that this oocyte is healthy and this one is stressed? On what basis? What kind of methods they used? Are the polscope data useful?
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In vitro culture conditions never meets the body environment (in vivo conditions). Therefore, once the oocytes are retrieved for IVF purpose, they are always exposed to stress conditions (due to minor changes in physical, temperature and culture medium). Oocyte is more susceptible to the stress conditions due to larger surface area (100-120 microM in dia) and even minor stress conditions may initiate downstream signaling pathways to induce oocyte death under in vitro culture conditions. We have identified some morphological features that could be used to identify the stressed oocytes in vitro. The series of morphological changes that are identified in oocytes cutlured in vitro are:
1. The smoothness (clear) of oocyte cytoplam is reduced
2. Initiation of cytoplasmic granulation
3. Changes in Zona pelucida histoarchitecture
4. Polar body roughness (fragmentation)
5. Cytoplasmic dia shrinkage (more gap between zona and corona)
6. Membrane blabbings
7. Cytoplasmic fragmentation
and many more........
For more details, kindly see few of our published papers:
1. Apoptosis, 10: 863-875. IF=4.677
2. Fertility Sterility; 84; 1163-1172. IF= 7.329
3. Free Radical Research, 42:212-220 IF=4.148
4. Free Radical Research, 43, 287-294. IF=4.148
5. Eur J Pharmacol. 667; 419-424. IF =4.432
6. J Biomed Sci. 23;36,1-5. IF = 8.410
7. J Biomed Sci 25(1);36 (1-7). IF= 8.410
8. J Biomedical Science, 26;11 (1-6). IF=8.410
9. Stem Cell Reviews and Reports, 17(3): 777-784. IF=5.739
Good luck
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I want to measure ROS synthesis in cells grown in transwell. I cannot measure fluorescence directly in the transwell because of the high so I have to remove the cells. One option is to use trypsin but I guess if someone has used another method. I use DCFDA kit (Abcam). Thank you
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If possible attach the cells on the coverslip. Once the cells are adhered on the cover slip, wash the cover slip three times and then expose to H2DCF (10 micro Mol) for 30 min in culture medium. After washing three times with culture medium, cover slip can be observed under epifluorescence microscope
using excitation at 480 nm, emission at 520 nm. The fluorescence intensity reflects the total ROS level in a cultured cell. You can perform the corrected total cell fluorescence (CTCF) analysis following the method published earlier using
Image J Software to analyze the total ROS level.
Let me clear you that this procedure will assess the total ROS level in a cell but not ROS synthesis. Good luck
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I treated SK-N-SH cells with 1mM H2O2 and 1mM H2O2+antioxidants (3 different mixtures) and measured the mitochondrial membrane potential (ΔΨm) with 500nM TMRE. As far as I know, H2O2 and ROS generation depolarizes the mitochondrial membrane so we expect lower values in TMRE signal rather than the H2O2+antioxidants mixure. I repeated the process 3 times and the result is the same.
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Dear Marc,
Dear Adam,
Before moving into mitochondrial membrane potential assays I measure the % viability in between Control-H2O2 treated cells and H2O2+antioxidants mixtures. The first group was significally moving downwards as time went by whereas the second one was almost near the control. Moreover, I meaured ROS production with DCFDA 5μΜ and results gave significant higher values in H2O2 group.
Cell do not detach but appear rather shrunk and morphologically changed.
I use Merck milipore hydrogen peroxide 30%
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Im trying to get Velocity in order to control underwater swarm ROV. I can use the acceleration and to Euler integration but the error will be accumulated over time. As we are having a swarm (many small robots) Im limited with sensors on board. So in order to get more accurate Velocity estimation was thinking of sensor Fusing of IMU and odometry. As don't have any odometry sensors came to some out of the box solution.
So as I have access to the motor (thruster- propeller) signals and the robot can move to maximum and constant speed. Means the rotation of the propeller have a maximum limit and when they reach that limit they can maintain that maximum. So was thinking of some mathematical model that can be used to provide me with odometry and than use some of the ROS packages for sensor fission(robot_lokalization or hector_pose_estimation) to get accurate Velocity
So I need a help in that mathematical model how to get odometry from the motors-thrusters. Any help with that and possible ROS node (C++) implementation?
Thanks
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Hi, could you teach me if anyone has experience in detection of Reactive oxygen species?
I would like to detect ROS in the cytosol and in the mitochondria separately.
Mitosox can detect mitochondrial ROS specifically.
I wonder that ROS detected by DHE is contained mitochondrial ROS?
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Here is an RG discussion that may help:
Some seem to use DCFDA with and without electron transport chain inhibitors. The use of DCFDA requires cautions as it is easily diffused from the cells as well as could respond to pH and other factors.
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I am working on lung inflammatory disorder which is marked by oxidative stress. In order to measure mitochondrial ROS in bronchoalveolar lavage fluid (BALF) sample procured from mice, I am planning to utilize mitoSOX probe. I have a query if we can directly use mitoSOX on BALF cells and measure the fluorescent intensity through flow cytometry? OR
Do we need to use antibodies (along with mitoSOX) against specific markers of inflammatory cells involved in lung inflammatory disease?
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BALF from infectious mice has both Macs and Neutrophils. So, If you want to compare ROS in a particular cell type, it is recommended to add a marker antibody in the experiment.
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To my understanding there is a kind of "black box" concerning the development of the disease itself: from its onset to the point when patients get hospitalized. Furthermore some potentially useful information coming from MDs is rarely taken into account. They often describe their patients as suffering from high altitude sickness. High altitude sickness is induced by a lack of oxygen.
During the first days of the disease the lungs are not yet damaged. The destruction of beta-hemoglobine has already been discussed, but this is seldom mentioned when we talk about different ethnic groups; modifications of hemoglobine protecting against Malaria might be the reason for their higher mortality.
A lack of oxygen is called hypoxia. Hypoxia induces the formation of ROS and RNOS. They can directly damage cells, amplify the inflammatory reaction and they act as second messengers in the immune system. It is even known they are related to human coronaviridae, but their role in COVID-19 is poorly taken into account.
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The COVID-19 infection causes hypoxia by depleting SpO2 level in patients. The hypoxia induces the generation of reactive oxygen species (ROS) . Another possibility is that the inflammation generated due to viral infection also increase ROS levels. The hypoxia-mediated increase of ROS further induce inflammation in the patients. In both conditions, ROS levels are elevated that further increase the severity of disease.
For more detail kindly see the published paper in Nat. Rev. Immunol. 20, 515-516 (2020):
References:
Laforge, M. Tissue damage from neutrophil-induced oxidative stress in COVID-19. Nat. Rev. Immunol. 20, 515-516 (2020).
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Hi, I'm trying to study the impact of reactive oxygen species on whole blood with respect to how it may induce proteolysis on an intracellular protein in PBMC's in a simulated extracorporeal circulatory model. Instead of exposure of PMBC's to hydrogen peroxide, I need to mimic the actual inflammatory environment such as cardiopulmonary bypass and therefore need whole blood. Trialed H2O2 diluted down to 0.05% without much success. Any suggestions would be much appreciated.
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It is not clear from your given details that which reactive oxygen species your are targeting for? For more focused suggestions, could you please make it bit more clear on your experimental targets? Anyway, with your given infromations, let me tell you that the hydrogen peroxide (H2O2) is a strong oxidizing agent and can generate OH- (hydroxyl ion) in a cell or solution. There are several reactive oxygen species and every one has its own pathway for damaging cellular histoarchtecture depending on the extent and nature of insult on the cellular/subcellular and macromolecules like protein and lipids. You can measure H2O2 level in blood serum/plasma using sensitive kits from R&D systems, USA. Total NO levels can also be measured accurately by using Nitrate/Nitrite kits in blood samples using kits from R&D systems, USA and other company make kits. Thus, you can analyze both H202 and total NO levels in blood samples.
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Hello everyone
I do not completely understand the theory of processing the data of flowcytometry DCFH-DA based ROS kit.
What do "Geo Mean" and "M2" refer to? Which one should I use as a ROS indicator?
Noting that they had given me conflicting results.
Any advice will be appreciated.
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Sorry for this late answer. The all means the total population count. It could be singlet cell, doublet cell or debrie. Of that, we only take or gate the cells, that are singlet and measure the DCFDA flourescence. Now, as the unstaind cells also have some autofluorescence, we have to first measure, the autofluorescence amount. On that plot or graph, we place a gate which will take those cells only which have fluorescence value, higher than the unstained value... This population is M2 population. In this M2 populatio, now you chech either your treatment increased or decreased the mean fluorescence intensity of the cells.
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Hi
I have IMU sensor that gives me the raw data such as orientation, Angular and Linear acceleration. Im using ROS and doing some Gazebo UUV simulation. Furthermore, I want to get linear velocity from the raw IMU data. If I do integration over time there will be accumulated error and will not be accurate with the time when for example the robot makes turns.
So If I use
acceleration_x = (msg->linear_acceleration.x + 9.81 * sin(pitch)) * cos(pitch); acceleration_y = (msg->linear_acceleration.y - 9.81 * sin(roll)) * cos(roll);
So integrating linear acceleration is very bad,
Velocity_x= Velocity_old_x+acceleration_x*dt;
because integrates the acceleration without taking into account any possible rotation of the sensor, which means that the results will probably be terrible if the sensor rotates at all.
So I need some ROS package that takes into account all this transformation and gives me the most accurate estimation of the linear velocity. Any Help? Thanks
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How to apply KF to the integration part? So, simple KF will reduce the error of the integration over time?
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I would like to use 2′,7′-Dichlorofluorescin diacetate to assess the ROS production in a549 cells growing in a 96-well microplate.
I have found protocols similar to the following in various papers, but I still have questions about the procedure.
  • Seed 5000 a549 cells by wells in a 96-well plate, incubate at 37°C overnight for adherence.
  • Preload the cells with 10 µm H2DCFDA in phenol red-free medium or in PBS for 30 min, 37°C in the dark (As I do not have a phenol-red free medium in stock, I would like to use PBS. But would it not be deleterious for the cells, or at least for their ROS metabolism, to be conserved in just PBS for such a long time?)
  • Remove the medium containing H2DCFDA and replace by a medium containing the ROS inductor (in my case, I would like to use H2DCFDA to study the impact of an ROS-inductor exposition for 48h. Considering the important time of incubation (48h), does a H2DCDA-loading after the exposition would not be preferable?)
  • Remove the ROS inductor containing medium and replace by 100 µl of PBS
  • Read the emission on a microplate reader (Ex/Em: ∼492–495/517–527 nm)
Any suggestions or advice would be welcome.
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Dear Professor Cepinskas,
Thank you very much for your suggestions. I will surely follow them during the optimisation process of this experiment.
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Reactive oxygen species was responsible for detection of Ascorbic acid, dopamine, uric acid and glutathione by colorimetric method?
In those case majority of nanozymes was OH radical and some super oxide anions. how selectively detection in the presence of ROS. the above mentioned biomelcules have selective shown in numerous papers but same reactive oxygen species? How that same ROS behave different for biomoelcule?
please suggest some valuable answer.
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Thank you your answers Mr. Shin Murakami and Mr. pradeep kumar
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I would like to measure mitochondrial ROS in different human fibroblast cultures, and I have in total 30 different cultures. Since superoxides are too easily converted, one tactic is to incubate one cell line, then measure. But with 30 cell lines, this would take quite a long time, and is the results then reliable. So the question is, can I set up let's say 5 cell lines, incubate with MitoSox, and then fixate the cells som how keep the fixed cell in the dark and then analyze? Does anyone have experience with this?
cheers,
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Please also have look at our Perspective article, if you like:
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Recently, I am performing the intracellular ROS detection by DCF-DA method in SHSY5Y cells through flow cytometer. But, I found a high fluorescence intensity for the untreated control stain with the probe DCF-DA at 10 micro molar for 30 min, 37 degree celcius in dark than that of the ros inducer i.e. rotenone. Why is it happening like this? Please tell me.
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Please also have look at our Perspective article, if you like:
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I have done simulation of single drone using hector quadrotor for indoor and outdoor environments using ROS kinetic and Gazebo7. Please help me to do the simulation for drone swarms using same package.
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is there hector_quadcopter repository available for ROS Neotic and gazebo 11? I wasn't able to find any or is there are any other quadcopter model to use with newer ROS Noetic version?
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Hi everyone. As GO or HA/FA itself produce ROS radicals if we treat them lonely with UV light but GO and HA/FA in NaCl suspensions do not produce ROS under UV light. What may be the possible reasons for it? Is quenching power of GO due to pie-pie stacking or competition of GO and HA/FA for ight absorption may be the leading mechanism for it? Or reduction of GO under UV promote the aggregation of GO due to which light absorption decrease and ultimately no ROS generation? Or there may be any role of NaCl?
Looking forward for a fruitful discussion.
Regards
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Thanks a lot sir Yuri Mirgorod for your kind response. Hope it will help me.
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We are looking to do a time dependent mtROS measurement after MPP+ treatment given to cells. Can anyone suggest if pretreatment with MitoSOX ( followed by washing) could be given before cells are treated with MPP+. I have found papers following this procedure for the total ROS estimation using DCFDA but couldn't find using MitoSOX.
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Thank you for your reply. However, the paper listed here has given treatment with MitoSox after 24h of MPP+ treatment.
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Hello everyone,
I am asking very general question, but i would appreciate if someone helps me. I want to use ROS for manufacturing process (Not robots) as a middleware that can create simulated Cyber physical system environment to enabling the interoperability of different nodes in real time. Before start involving in ROS, i would like to ask you below questions.
  1. ROS can be usable other than robot application for our manufacturing system ?
  2. If yes, then what challenges i will face? if i want to use it for manufacturing system?
  3. What are advantages/disadvantages of using ROS as a middleware in normal manufacturing process?
  4. How much time it will take to develop ROS middleware for normal manufacturing process? (I never used ROS)
  5. I know C++/Python are good options, but what programming language is best for me?
  6. Is their any other options are available other than can use as a middleware in my manufacturing process that can create simulated Cyber physical system environment to enabling the interoperability of different nodes? I would appreciate if someone suggest me other options, which can easy to use than ROS.
Thanks a ton
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Hi.,
The Robot Operating System (ROS) is a flexible framework for writing robot software. It is a collection of tools, libraries, and conventions that aim to simplify the task of creating complex and robust robot behavior across a wide variety of robotic platforms.
robotics projects
  1. ROS is general. ...
  2. ROS packages for everything. ...
  3. ROS is language-agnostic. ...
  4. ROS has great simulation tools. ...
  5. You can control multiple robots with ROS. ...
  6. ROS is light. ...
  7. More and more compatible ROS products. ...
  8. ROS is an open source project with a permissive license
ROS is an open-source, meta-operating system for your robot. It provides the services you would expect from an operating system, including hardware abstraction, low-level device control, implementation of commonly-used functionality, message-passing between processes, and package management.
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I want to determine ROS levels in freshly isolated and/or snap-frozen primary leukemic cells. Any idea? Please suggest references, if possible.
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I doubt you could assess DCFDA in frozen cells but here are some refs:
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Is there any photosensitizer that can generate different types of ROS upon different light irradiation? I understand my question is kinda confusing so I added an example below this.
(E.g., When the photosensitizer is irradiated by white LED light, it generates singlet oxygen mostly. On the other hand, when it is irradiated by green LED light, it generates radicals mostly.)
I want to find something about detection of ROS content produced by different light irradiation to the photosensitizer.
If you guys can find any journals that can answer my question, pls let me know! thank you.
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Photosensitizer based Journal about conversation and storage
International Journal of energy research, International Journal of Electrical power and energy system, energy source A,etc.
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ROS (reactive oxygen species, DCFH-DA, DHE), TAC (Total antioxidant capacity). ROS measured using flowcytometry but TAC measured by a kit and elisa reader.
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Not necessary ever, but if you found a significant difference by using the ratio you can include it .
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I am treating my cells with a drug that generates about 20-40 uM H2O2. I see a profound activation of gamma-H2AX but no/very low levels of corresponding DNA damage in comet assay.
I am trying to find out if the induction of gamma-H2AX is somehow related to the generation of H2O2 in my drug treated cells.
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Thank you so much!
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Hello
It is been a long time I have been trying to stimulate vascular smooth muscle cells (MOVAS) for ROS generation. I am using DCFDA assay for ROS detection....
Everything I tried, has failed to stimulate it! LPS (short and long incubation time), high and low concentration!, Iron (Fe3+), glucose (25 mM 50 mM etc), Beta-glycerophosphate (up to 10 mM).... none works!
Does anybody have any idea what is happening?
Extremely frustrating cycle of trial and failure !!!
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If you need a positive control, just treat them with H2O2. Otherwise nicotine or high calcium (3.6 or 5.4 mM) worked in my hands, in primary human vascular smooth muscle cells.
The problem might be in the MOVAS cells themselves? Maybe the fact that it's a cell line makes them insensitive to stimuli.
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I used MitoSox red dye for the measurement of ROS levels in pancreatic cancer cell lines.
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Thank you so much Denis and Luiz
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I'm working on cell apoptosis now, and I really need a stable inducer of intrinsic apoptotic/ROS-related/mitochondria-related apoptosis. I've tried H2O2 and ABT(chemical drugs), but they both have unstable results. So if anyone has any suggestions, it would be very nice.
Thanks :)
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Thank you all for the help!
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Does anyone have an idea related to commercial availability of any NIR probe for in vivo detection of ROS?
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How are Nir probes used to detect mitochondrial co?
The other is that the probes for CO display excitation and emission within the ultraviolet or visible range, which hinders their applications in vivo. Herein, a hemicyanine-based near-infrared (NIR) fluorescent probe named CyAPC is first synthesized and used to detect mitochondrial CO.
Mitochondria-targeted near-infrared fluorescent probe for ...
How to imaging ROS production in Drosophila in vivo?
Here, we describe a protocol for imaging ROS production in Drosophila using either H2DCF or DHE. We highlight the specific advantage posed by monitoring ROS production in vivo by comparing the phenotype of cells mutant for genes encoding mitochondrial proteins with their wild-type neighbors.
Scientific Protocols - A protocol for in vivo detection of ...
How does near infrared ( NIR ) fluorescence imaging work?
Near-infrared (NIR) fluorescence imaging is a noninvasive technique that provides numerous advantages for the real-time in vivo monitoring of biological information in living subjects without the use of ionizing radiation. Near-infrared fluorescent (NIRF) dyes are widely used as fluorescent imaging probes.
Research progress of near-infrared fluorescence probes ...
How are ROS levels detected in the body?
Thus detection of ROS levels has relied largely on detecting end products – either by chemiluminescence or by fluorescence – that are formed when specific compounds react with ROS (3 4).
Scientific Protocols - A protocol for in vivo detection of ...
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In Egypt, ambient temperature can remain consistently high for extended periods of time and sudden recurrent hot and humid waves that have more harmful effects. So poultry production suffers significant losses every year because of heat stress, leading to economic losses to the poultry farmers. During these periods temperatures approached 40˚C most of the time and humidity reaches 75%. A temperature above 30˚C represents a heat-stressed condition for birds and is one of the most common stressors that affect poultry production criteria where the ideal temperature for broilers is 10-22˚C to get optimum body weight and 15- 27˚C for feed efficiency.
Heat stress is principally important in intensive poultry operations especially in broilers lines because their higher production performance and feed conversion efficiency make today's chickens more susceptible to heat stress than ever before.
High mortality, decreased feed intake, lower body weight gain and poor feed efficiency are common adverse effects of heat stress often seen in meat-type poultry flocks. In addition, heat stress increases lipid oxidation as a consequence of increased free radical generation, a condition that enhances the formation of reactive oxygen species (ROS) and induces oxidative stress in cells. Antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) play a vital role in protecting cells from the harmful effects of ROS. Synthesizing these enzymes is an important regulation, in terms of animal response to stress conditions. However, this response will be effective only if cofactors such as Se for GPx and copper, zinc, and manganese for SOD are available.
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Heat stress is divided into acute heat stress and chronic heat stress with the participation of different antioxidant enzymes. Under acute heat stress, ROS level in the body is rapidly increased and the antioxidant enzyme system also responds rapidly, by which the activity of CAT, SOD, and GSH-Px is increased significantly to remove excessive free radicals. Chronic heat stress can break the antioxidant enzyme system and cause ROS accumulation in the body to induce oxidative stress by decreasing the activity of CAT, SOD, and GSH-Px.
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I am trying to detect ROS in some breast cancer cell lines, including MDA-MB-231, MCF-7 and 4T1. I found out that ROS in some cell lines decreased after the incubation with H2O2, while in some cell lines ROS increased. I used DCFH-DA as prober. I just feel confused about the results. Could someone provide some clues?
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I have worked on the effect of some extracts on the inhibition of ROS, similar to what you mention, in my case, the extract could inhibit ROS compared to the sample that was not treated with the extract, i.e. it could decrease the oxidative stress in the cell. But I have also read articles in which they work with cell lines, in which they try to provoke this oxidative stress in the cells to induce apoptosis. But as mentioned in the previous answer, it is not possible to know what specific ROS you are measuring.
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I used DCFH method to measure reactive oxygen species in the atmosphere, using hydrogen peroxide as the standard solution, and found that when hydrogen peroxide is not added, the fluorescence intensity is very large, I do not know what is the reason?
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DCFH is known to react with a bunch of molecules other than H2O2 or reactive oxygen species.
Take a look at:
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I am interested in determining the intracellular ROS concentration but we do not have a fluorescence spectrophotometer. Could someone recommend an alternative technique where the determination is carried out in UV-visible and is adaptable to a 96-well plate.
Thank you
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DOI: 10.1179/135100009X392557
This article may be of your help. ROS was measured at 505 nm.
All the best.
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Hi!
I am looking for a fluorescente probe to evaluate ROS levels in specific subregions of mouse brain. I tested DHE but the staining was totally unspecific!!!
To test DHE before the experimental conditons, I tested the compound in acute brain slices treated with Rotenone: 2um, 1um, 500nm, 200nm (3min) followed by 15 min wash.
The control slice was not treated with Rotenone.
I fixed the slices in PFA 4% over night, sunked in saccarose 30% and the cutted in 30um slices.
DHE staining protocol:
-staining: 5um, 15', RT
- wash: PB, 2x1min
- wash: H20, 1min
Unfortunatelly, all neurons of all conditions (also the control one!) showed high fluorescent signal!!!!
Thus, I stained 30um slices from a perfused brain (from naive mouse) and I checked an intense and unspecific fluorescent staining in all neuronal population also in this condition!
How can I solve this problem?
Can someone suggest me a different probe to analyze ROS in mouse brain?
Thank you for your help!!!
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Please try the protocol described in this ms:
Peterson SL, Morrow D, Liu S, Liu KJ. Hydroethidine detection of superoxide production during the lithium-pilocarpine model of status epilepticus. Epilepsy Res. 2002 May;49(3):226-38. doi: 10.1016/s0920-1211(02)00047-5. PMID: 12076844.
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I would like to label some fixed cells/platelets with CM-H2DCFDA (General Oxidative Stress Indicator) dye . I would like to know if this is possible in fixed cells. Does the dye bind covalently to intracellular targets? Do fixed cells even have ROS?
According to ThermoFisher Scientific:
CM-H2DCFDA is a chloromethyl derivative of H2DCFDA, useful as an indicator for reactive oxygen species (ROS) in cells. This indicator exhibits much better retention in live cells than H2DCFDA. CM-H2DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell, thus facilitating long-term studies.
Thank you.
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CM-H2DCFDA dye was used for analysis the oxidative stress of cancer cells after drug treat. In here we did not fix the cells. Analysis with FCM. In cell cycle analysis cells was fixed with 70% ethanol.
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I incubated E.coli (10^8 CFU/ml) with DCFH-DA (final concentration 1 mM) in dark for 30 mins. Then I scanned the sample for an excitation wavelength of 485-495 nm and an emission wavelength of 500-600 nm. But I did not find any significant emission peak (as shown in the attached figure).
Please advise me on how I can improve the ROS detection by DCFH-DA.
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Dear Marc Herb,
Our microplate fluorimeter is out of order. We have a microplate reader which can only measure absorbance, not fluorosence. I also want to try microplate reader. For that I need to take my samples to other department's lab. Now my question is that how long the samples remain stable after incubation with DCFH-DA if I keep them in -20?
Thanks a lot for your suggestions and insights.
Regards,
Shayok
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My research is looking to detect cell activation (specifically PAMs) through ROS measurement.
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Dear Claudia,
CellROX oxidative stress reagents are different cell-permeable fluorescent probes that are mainly used as indicators for oxidative stress. The different CellROX probes are diffusible and cannot differ between cellular compartments and ROS subspecies, but vary greatly in usage conditions, like live cell compatibility, resistance to detergents, fixation capabilities and emitted fluorescence. CellRox Red is not resistant to detergents while CellROX Green is resistant
If you like, have a look at the pdf of the distributor:
if you like, you can have also a look at our review:
It also shows the mistakes people make with interpreting ROS measurements, when using wrong ROS probes or ROS scavengers.
If you have any further questions please ask any time.
All the best and stay healthy,
Marc
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Hallo everyone,
I am trying to decrease ROS by adding glutathione (reduced) in human retinal endothelial cells under hyperoxic conditions. Unfortunately a lot of cellular ros assay kits like H2DCFDA are based on fluorogenic dyes which react with glutathione. Does anyone have experience or an idea how to measure oxidative stress and/or ROS based on reactions without interference with strong ROS scavenger like glutathione?
Thank you in advance
Justus
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Justus S. Ernesti came across this paper for your question, though not the accurate one but to give you an idea
srinivas kasulla
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I`m trying to measure increased ROS levels, caused by ATP in RAW macrophages, but I see no difference versus control. I used HBSS, 300 μM - 3 mM range of ATP and 3 hours - time of incubation. After that I included LPS priming (100 ng/ml). It makes result only with 100-300 μM, but I can`t get good repeatability and cell viability (cells stop adhering and are washed out of the plate). Сan anyone advise a suitable protocol?
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Hey Sergei,
that sounds like a lot of problems in this assay. First of all, when your cells start detaching, then there is no chance of getting reliable ROS measurements in a plate reader. Either you switch to FACS or you have to switch or adapt conditions (ATP/LPS duration and concentration), that do not drive your cells into cell death. It is not the HBSS. We cultured macrophages (PM or BMDM) with HBSS at least for 5 hours, and they where fine and not detaching.
If you get that setting right, the next question is, what ROS probe did you use? Not every cell produces ROS in every compartment with every stimulus. So there is a good chance that your cells either do not produce ROS at all after your stimuli or you miss your ROS production because you look at the wrong compartment.
People tend to just increase their stimuli until "they see some ROS production", but in my opinion that is not accurate. If you see with your stimuli reactions of your cells in other assays, but no ROS production, then these stimuly might just not induce ROS production.
Also the view of ROS "freely diffusing" through the cells once produced is, in terms of the highly regulated redox balance in each compartment, just bullshit.
We have established ROS measurements in macrophages and we are using our protocols for several years with great success. If you like you can have a look at our papers, how ROS measurements look in a plate reader:
If you like, you can have also a look at our review:
It also shows the mistakes people make with interpreting ROS measuremtns, when using wrong ROS probes or ROS scavengers.
If you have any further question, please feel free to ask any time. I am happy to help.
All the best and stay healthy,
Marc
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Dear all,
These days I had a problem in choosing the mode of microplate mode. I need your help to know if what I did is right.
My goal: To use MitoSOX (Ex/Em: 510/580 nm) to measure mitochondrial ROS in macrophages.
You can find Structure & Spectrum here https://www.bioblast.at/index.php/MitoSOX
"The fluorescence intensity of MitoSOX was measured by a microplate reader...at excitation and emission wavelengths of 510 and 580 nm, respectively."
So, I switch on my lovely Tecan plate reader, open "Magellan" software, chose "Fluorescence Intensity" ----"Wavelengths"...then...I chose "Fixed Wavelength" mode and set the "exciation wav" and "Emission wav" into 510 and 580. but...Is this right?
By the way, in which condition do you recommend "excitation scan" mode or "emission scan" mode rather than "Fixed Wavelength" mode ? As a beginner, I really do not know the difference now.
I summarised my setting in the attached jpeg and pptx.
Thanks.
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Hello
You are right. Since you know the excitation and emission wavelengths you should select the 'fixed wavelength mode'.
In excitation scan mode you select the wavelength which gives the maximum absorbance as your excitation wavelength.
In emission scan mode you select the wavelength that gives the maximum absorbance as your emission wavelength.
So the two best wavelengths would be those that produce the maximum signal in the excitation and emission spectra
that is produced during excitation scan mode and emission scan mode respectively.
These scan modes are used when one does not know the excitation and emission wavelengths.
I hope this is helpful.
Regards.
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I have exposed microalgae cells to temperature stress and measure the ROS level using DCFH, but I found that the DCFH was significantly lower in stress-treated samples compared to control. How is this possible?
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Dear Bahram Barati,
There are chances of decreased expression of ROS in the system under the stress condition if this is the result of the repeated standardized experiment.
But, decreased expression of ROS also tagged with other parameters such as involvement of various antioxidant enzymes, the incubation time factor, methodology of the experiment, etc.,
you can run parallel experiments regarding the antioxidant enzyme studies and GSH in order to confirm with the obtained result (decreased ROS in stress condition than Control).
All the best
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