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Oxidative Stress Biomarkers - Science topic

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As a Biomarkers, I have protein concentration, protein carbonyl content, and GST activity in tadpoles. From this data result, I prepared plots and now I want to explain the relation between them. Does anyone know how they are related to each other?
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Thank you Azadeh Eshraghi for your answer.
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I'm looking for laboratories working with oxidative stress to test nitrocellulose redox permanganometry (NRP). NRP is a simple method for reductive capacity assessment based on the analysis of the MnO2 precipitate following redox reaction between KMnO4 and biological samples fixed onto the nitrocellulose. This method estimates how well the sample can give up its electrons to KMnO4, so it estimates the amount of chemical antioxidants. In other words this method can be used to measure total chemical antioxidant capacity of biological samples. Advantages of the method are: great precision and accuracy; the possibility to measure a lot of samples simultaneously; simplicity; cost (you only need a piece of nitrocellulose membrane and KMnO4). Moreover, the method can be used to obtain information on the spatial distribution of antioxidant capacity in tissue sections (both cryosections and FFPE can be used), providing unique information on the redox status of the tissue.
As the method is new, and we proposed it just recently, I'm looking for laboratories that are working with oxidative stress to test the method in their samples. We conducted thorough analyses and the method seems to be very robust. Nevertheless, I believe additional independent testing is always a good idea. Also, I believe comments of researchers closely working with oxidative stress would be beneficial for further development of this simple method.
The original paper can be found here:
A step by step protocol can be found here:
Best,
Jan
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In my opinion, there are no simple methods to evaluate antioxidizing capacities of potential antioxidants. General tests like DPPH, ABTS... only (roughly) measure the ability of the antioxidants to scavenge free radicals. An antioxidant is much more than that; it can act as a scavenger of free radicals, repair of the oxidized species (reduction by one electron transfer) or by cascade (cumulative) effect . Furthermore, the antioxidant capacity can also depend on the agent responsible for the oxidative stress conditions (selective antioxidants).I recommend the evaluation of the mutual behavior of both antioxidant and the target to be protected in every specific oxidizing environment.For details see e.g.:
  • Santos, P.M.P.; Telo, J.P.; Vieira, A.J.S.C. "Structure and Redox Properties of Radicals Derived from One-electron Oxidised Methylxanthines", Redox Report (2008), 13(3), 123; DOI: 10.1179/135100008X259231
  • Santos, P.M.P.; Antunes, A.M.M.; Noronha, J.; Fernandes, E.; Vieira, A.J.S.C. "Scavenging activity of aminoantipyrines against hydroxyl radical", Eur. J. Med. Chem. (2010), 45, 2258-2264; DOI: 10.1016/j.ejmech.2010.01.071
  • Santos, P.M.P.; SILVA, S.A.G., JUSTINO, G.C.; VIEIRA, A.J.S.C. "Demethylation of Theophylline (1,3-Dimethylxanthine) to 1-Methylxanthine: the First Step of an Antioxidising Cascade", Redox Report (2010), 15(3), 138; DOI: 10.1179/174329210X12650506623726
  • SANTOS, P.M.P.; VIEIRA, A.J.S.C.Antioxidising activity of cinnamic acid derivatives against oxidative stress induced by oxidising radicals”, J. Phys. Org. Chem. (2013), 26, 432; DOI: 10.1002/poc.3104
  • VIEIRA, A.J.S.C.; SANTOS, P.M.P. “A Tentative Classification of Antioxidants: Which Role They Play when Protecting Biological Targets from Oxidative Stress Induced Damage?”, J Med Chem Drug Des (2017), 1, 1-3; DOI: 10.16966/jmcdd.101
  • VIEIRA, A.J.S.C.; GASPAR, E.M.; SANTOS, P.M.P. “Mechanisms of potential antioxidant activity of caffeine”, Rad. Phys. Chem. (2020), 174, 108968; DOI 10.1016/j.radphyschem.2020.108968
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How can i optimize the protein amount obtained in cardiac homogenates when working with limited amounts of tissue (mice)? (Homogenates will be used to measure oxidative stress biomarkers)
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Spectrophotometric methods to optimize the amount of prote have been investigated in the following article
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Tobacco plants were subjected to 3 different nutritional treatments (A, B, C) for 3 weeks, and also were treated under two irrigation regimes during 2 weeks (Well-watered plants or Control plants, and Water deficit treated plants or drought plants). During drought plants treated with the treatments called "C" showed: (i) better water parameters, (ii) higher growth than control, (iii) higher water use efficiency and water saving, (iv) better recovery from extreme drought, etc.
Updated information:
  1. Model organism: Tobacco (Nicotiana tabacum L.)
  2. Pot size: 7.5 L pots (pot size 20 cm × 17 cm × 25 cm)
  3. Substrate: a mix of perlite:vermiculite (4:6)
  4. Treatments: Seeds were sown and two weeks later (15 days after sowing, DAS), seedlings were transplanted to 7.5 L pots. Then, plants were subjected to three different nutritional treatments. After 30 days (45 DAS), in addition to the three nutritional treatments, plants were also subjected to two irrigation treatments: optimal irrigation (control; CTR), in which pots containing tobacco plants were irrigated up to 100 % field capacity (3.5 mL g-1 substrate) throughout the experiment, and moderate sustained water deficit (WD), with pots irrigated every two days up to 60 % of field capacity (2.1 mL g-1 substrate) for 20 days (64-65 DAS).
Fresh biomass was collected in each treatment, and the following organic compounds were determined: MDA, H2O2, PROLINE, and PHENOLICS. Also, the PEROXISOME CATALASE activity was determined.
-Malonyl Dialdehyde (MDA) content, hydrogen peroxide content, and catalase activity are cellular oxidative stress biomarkers.
-Proline is a very important amino acid that which accumulation is correlated to plant stress tolerance
- Phenolics (phenolic compounds and flavonoids) are the largest group of phytochemicals that account for most of the antioxidant activity in plants.
In the attached figure there are the results of the ANOVA statistic in CTR or DROUGHT plants, and also is showing the logarithm with the base of 2 of the ratio between drought and control values to understand the decrease or increase in drought plants, in contrast, to control plants for each parameter.
What I see in this result is that there is no clear pattern related to a water deficit regarding the great results obtained in water parameters, plant biomass, water consumption/efficiency, photosynthetic activity, recovery from water stress deficit, etc.
To sum up there's complete nonsense in results in contrast to "C" treatment:
-No changes in MDA (reduction in the other nutritional treatments???????)
-Increase in CAT y H2O2???????
-a decrease in PROLINE????
-An increase in PHENOLIC compounds is logical due to the increase in H2O2, but it has no sense in plants that are more tolerant to drought.
I hope that someone might help me resolve this nonsense
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Hello J.D. Franco-Navarro
Your question can’t be answered as @Fahad Shafiq has correctly pointed out at the end of the comments because there is almost complete lack of information about the methods. The study seems to have been done with rather small plants, and in such a way that there would be a strong interaction between nutrition and water supply, making interpretation very difficult. Let us assume you grew the plants in soil, applied a liquid fertilizer and allowing some plants in each nutrient treatment to dry by stopping watering, while the control was given ample water. That is a standard molecular biology/biochemistry approach to such studies.
Which was the treatment supplying the most nutrients - A I suspect, with C supplying least. So plants in A would grow better than in C, and have more, larger leaves. When you started the drought treatment plants in A would dry the soil much faster than those in C – simply a matter of greater surface area in A. So plants in A would become severely drought stressed before those in C, which would appear to grow and be much more `drought resistant’. They would also have very different contents of metabolites. The lesson from this – if my supposition is true - is that it IS ESSENTIAL TO COMPARE PLANTS AT THE SAME RELATIVE WATER CONTENT. If this is not done then the study is invalid. I suggest my paper explains the problem and ways of doing such studies correctly – it focuses on GM plants but the problem is absolutely the same for all studies involving water supply.
Lawlor DW. Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities. J Exp Bot. 2013 Jan;64(1):83-108. doi: 10.1093/jxb/ers326. Epub 2012 Nov 16.
By ignoring the complexities involved with water supply in solid matrices and the effects of different rates of water loss, studies of the metabolism, genetic modifications etc are invalid. Hope this rather strong critic does not apply to your study. Please provide the necessary information about methods.
Best wishes
David
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1- what is the best OS marker to evaluate oxidative stress statue in a research study in a pre-diabetic/ diabetic population? what markers are more reliable for an academic-clinical study?
2- to have enough evidence on oxidative stress statue, how many markers should be measured? is measuring one or two marker enough to get a reliable result?
3- if it were you, which two would you chose from the following list for a clinical study?
total antioxidant capacity, SOD, GSH-Px, Malondialdehyde.
4- Malondialdehyde is a frequently used marker, but it isn't very specific. Is it still a good marker to use (ELISA method)?
best regards
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Possibly changed parameters are GSH, and a ratio of GSH:GSSG, SOD, MDA, plasma antioxidant vitamins, GPx and catalase.
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Dear colleagues;
Are reduced glutathione (GSH), uric acid, and vitamin C biomarkers for oxidative stress in biological systems?
Please, provide your answer with reference/s
Regards
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Thanks Dr. Luay & Dr. Sarah
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Dear colleagues;
What are the modern protocols to measure GSH, GSSG, SOD, MDA, catalase, and GPx?
Please support with ref
Best regards
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I get bad experience about using Spectrophotometric analyses in MDA, GSH and antioxidant vitamins
So I didn't agree in past with colleagues who suggested the SPA
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I have struggled with the MDA assay for a while now. My samples are SHSY-5Y neuronal cell lines and i treat them with 500 uM hydrogen peroxide, 1.5 mM potassium bromate and 500 mM Ethanol to induce oxidative stress. I have tried the 2 methods attached, the first being a sigma assay kit yet the standards seem to be fine as there is the development of the pink colour and a nive standard curve afterwards but no reaction so far have been noticed in the treated samples. Any suggestions from previous experiences? or any proven alternative method?
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I also have a same problem with plasma and serum samples. Some of the samples react to produce color while others failed to develop color. I could not understand why it happens.
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What are the standard units for Urinary Malondialdehyde relative creatinine?
Is it
umol.g creatinine?
umol.mmol creatinine?
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Mediators of Inflammation Volume 2015, Article ID 524291, 6 pages http://dx.doi.org/10.1155/2015/524291
Research Article
Urinary Malondialdehyde Is Associated with Visceral Abdominal Obesity in Middle-Aged MenUrinary
MDA was measured with high performance liquid chromatography (HPLC). Urinary MDA levels were expressed as μmol/g creatinine, averaged, and used for analysis.
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Hello,
I am working on a project measuring a number of oxidative stress biomarkers in the urine of clinical samples. We are particularly interested in 8-OH-dG and f2isoprostanes, but we are having problems with the sensitivity of our method. We cannot get anywhere near reported literature measurements for these markers using a Thermo TSQ Vantage. Has anyone used the Vantage for these markers in urine successfully? I am a clinical researcher, but can easily get details on the LC-MS method from our collaborator if there are suspicions of something being up with the actual method (although we suspect it is machine related).
Thanks,
Daniel
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Hey Harald,
We are trying to both. 8-OH-G appears to be fine, but we really wanted to get the 8OHdG and F2ISO in the same run. May have to do a couple runs in the end. Thanks for the suggestion.
CHeers,
Daniel
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I'm planning on looking at oxidative stress markers in quail red blood cells we have stored at -80.  I'm planning to run them on an ELISA measuring protein carbonyls and SOD concentrations among other things.  
I would like to know if anyone has managed to run similar techniques on frozen RBCs as opposed to freshly prepared blood cells and if they encountered any issues or offer advice on how to prepare them correctly.
Many thanks,
Dave
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Hi David, I am working at the University fo Glasgow on Japanese quail too, with a main focus on oxidative stress. Please send me an email if you want more info, but for methods to measure oxidative stress from red blood cells, you can have a look at most of my papers on RG. I measured protein carbonyl, DNA damage, SOD, GPx, glutathione (total and oxidized).
All the best,
Antoine
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Hi every one I need more information about how low temperature can affect oxidative stress parameters in fresh water fish
 bet regards
Safia Habila
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Interesting question Safia..
Extreme Temperature Responses, Oxidative Stress and Antioxidant Defense in Plants (DOI: 10.5772/54833)
The cellular changes induced by either high temperature  or low temperature  include responses those lead to the excess accumulation of toxic compounds, especially reactive oxygen species (ROS). The end result of ROS accumulation is oxidative stress]. In response to high temperature  the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) can lead to the production of H2O2 as a consequence of increases in its oxygenase reactions . On the other hand, LT conditions can create an imbalance between light absorption and light use by inhibiting the activity of the Calvin–Benson cycle. Enhanced photosynthetic electron flux to O2 and over-reduction of the respiratory electron transport chain (ETC) can also result in ROS accumulation during chilling which causes oxidative stress. Plants have evolved a variety of responses to extreme temperatures those minimize damages and ensure the maintenance of cellular homeostasis. A considerable amount of works have explored that there is a direct link between ROS scavenging and plant stress tolerance under temperature extremes . Thus, the improvement of temperature stress tolerance is often related to enhanced activities of enzymes involved in antioxidant systems of plants. Plants exposed to extreme temperatures use several non-enzymatic and enzymatic antioxidants to cope with the harmful effects of oxidative stress; higher activities of antioxidant defense enzymes are correlated with higher stress tolerance. Different plant studies have revealed that enhancing antioxidant defense confers stress tolerance to either  high temperature  or low temperature  stress .
Like annual crops, perennial crops are also sensitive to extreme temperature. Fruits and nut trees are important crop plants which often face extreme temperature stress induced damages. Every fruit tree species has a range of optimum temperatures  above or below which the growth and yield markedly reduced. The mean temperatures range for optimum growth of most tropical fruits are about 24-30°C . For instance, mango (Mengifera indica) tree can tolerate high temperature up to 48°C only for a certain period of time , on the contrary it has only partial tolerance to low temperature  In another study, Schaffer et al. observed that monoembryonic mango cultivars tend to be more low temperature  tolerant than polyembryonic cultivars . However, several studies have shown that low temperature promote reproductive morphogenesis in mango. Dinesh and Reddy studied the responses of fruit trees to temperature and observed differential responses to temperature in different fruit species. They concluded that lychee and longan require a warm sub-tropical to tropical climate that is cool but also frost-free or with only very slight winter frosts not below -4°C, and with high summer heat, rainfall, and humidity. In longan, stressful temperatures of <15°C at the young fruit stage reduce fruit growth potential and final size as reported by Young et al.. Stressful  low temperature  also induces excessive fruit drop and severe fruit cracking ....
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I am looking for a way to use fluorescent microscopy in order to measure the levels of hydrogen peroxide in C. Elegans which exhibit Alzheimer’s symptoms like neurodegeneration.  It is ok if there are two separate strains which specifically have some sort of marker for either hydrogen peroxide, catalase, or oxidative stress.  Also, if you mention any strains, please link the article from which it is from.
Thanks in advance
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Dear Mir Alam,
Oxidative stress can be measured using any C. elegans strain. Our group uses DCF-DA as a fluorescent probe to measure ROS accumulation, not specifically H2O2. I just tried the previous method for N2, CF1038 and GR1307, but I guess it would also work for Alzheimer’s-related-strains, such as CL2006, CL4176, etc.
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In any treated animal group if we observe a significant increase in a number of parameters, for example oxidative stress measuring enzymes like superoxide dismutase, GSH, catalase or lipid peroxidation. Does this increase simply should be worrisome about the toxic potential of treated chemical or preventing from its exposure.
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In either case , it is important, if there is a significant difference or no significant difference, important is to have scientific reasons to justify either of cases.
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Hi experts,
I need your help:
What is the best protein (antibody) marker to measure oxidative stress/reactive oxygen species using western blot or qPCR?
I have seen SOD2, Nox2/Nox4, NOS, or catalase in different publications? My personal/logic choice is SOD2 since it is directly involved in the catalytic reaction for H2O2 production, but may not be a direct marker of oxidative stress. So I am also considering catalase... I need your opinion, which one do you think is the best? And also, I found in another thread, is it true that this is not a good way to measure oxidative stress from brain tissue extracts? According to others' comments, paraformaldehyde can affect the protein and consequently the catalytic activity, which seems logical.
Thanks for your answer.
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Any of the ROS/RNS kits should work as well as DCF. Another good indicator of OS would be oxidized and reduced glutathione levels (GSSG/GSH)
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I am using NMR to determine levels of ROS from serum that is currently stored at -80, and I would like suggestions for the best biomarker to measure.
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Thank you Mukesh,
I will check glutothione peroxidase can be done on NMR,
Katie
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I have the 1 ml method , but I have a small amount of plasma and I need a micro method.
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Gabriela, see in the attached file a paper that we published. 
Best regards
José
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I am performing the SOD assay using the pyrogallol protocol by Marklund and Marklund for estimating SOD from serum. I am not getting the desired results. I'm using a 30 mM concentration of pyrogallol, but my reading of serum is more than the pyrogallol reading.
Can anyone suggest any solutions.
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I think the important aspect is not comparing the OD between control (Pyrogallol) and samples. It is obvious that the OD of samples will be greater than that of the control. The important thing is looking at the mean difference per minute. If still the mean difference of samples are still higher, then try to look at your buffer (Tris 0.05M EDTA 0.001M pH 8.5). You can try 20mM pyrogallol.
Best regards
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Hi Everyone I am looking for antibodies for ER pathways that works in INS-1 cells or any other cells. There are lots of paper out there but I want to know if anybody have tried these antibodies in INS-1 cells and that has worked perfectly. The antibodies I am interested are as follows:
spliced-XBP1. p-PERK, PERK, p-IRE1 alpha, Total-IRE1 alpha, ATF4, ATF6 active.
Any information will be highly appreciated. Thanks in advance.
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UPR/ER stress is activated through an orchestrated interplay of signaling molecules from all three arms of the UPR pathway i.e. IRE1, ATF6, and PERK. This is why, it is critical to analyze signaling players in the context of the mentioned fact. Novus' ER Stress / UPR Antibody Pack contains sample size vials of the top markers namely: GRP78/HSPA5, pSer724 IRE1 alpha, total IRE1 alpha, XBP1, ATF6, and CHOP/GADD153. For scientific technical answers to over 15 similar questions, I would suggest reviewing our UPR and ER Stress FAQs at https://www.novusbio.com/support/upr-and-er-stress-faqs
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I am looking for the gene sequence of above mentioned genes. Plz send the gene sequences Or else plz suggest me the way to search the gene sequences. 
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Thank You sir (Mr. Vrajesh Patel). I will al ways be grateful to you.
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I am finding mitochondrial oxidative stress marker in tissue or analysis kit.
I am using 8-OhdG antibody for detecting MtDNA oxidative damage. But I need more additional data to advance the result.
Can anyone tell me mitochondrial oxidative stress marker in tissue ?
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Thank you for your helps.~!
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I have luminol and a spectrophotometer (and basic lab equipments and instruments), nothing more. My treatment (Hydrogen peroxide) will cause production of ROS inside the HEK293 cells and I would need to be able to measure this. My specific questions:
a) Is it possible to make a standard curve for ROS concentration using Luminol? How?
b) Could you point me to papers that might have done this before?
c) Is the concentration (in mM, say) of ROS actually measurable (directly or indirectly)? 
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Luminol can be measured by chemiluminesence and gives both extracellular and intracellular ROS. Lucigenin is another probe. 
Similarly you can try to use DHE and DCF but these are sepcific for either superoxide or hydrogen peroxide, whilst Luminol will measure all ROS.
The way in which you will have to quantify it mught be challenging. Perhaps express as per number of cells (e.g. relative light units per million cells or absorbance per number of cells.
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I have been recently trying to study mitochondrial structures in our cell line B16. Previously we used the Mitotracker Deep Red dye for this study and it worked fine. But in the last few experiments I have realised that staining with Mito deep red for even 20min @500nM concentration in both live and fixed cells led to a total degradation of the mitochondria. I have done parallel staining of experimental duplicates with Mito tracker Green dye and checked that mitochondria in these cells are in good condition. It is only when I add Mitotracker Deep Red do I see a degradation. Has anyone faced anything similar or could give some explanation for this problem as I am not able to solve it myself.
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The regular dose of MitoTracker Deep Red is 25 nM (50 nM already gives near saturation fluorescence), with 30-min incubation at 37C. 
Your mitochondria (live) may have been overloaded. In fact all mitotrackers (green, red) work around this dose range, if your microscope lasers are efficient enough. 
**
As for staining fixed mitochondria, it is not recommended based on chemical knowledge of the tracker structures. All commercial mitochondria trackers carry a positive charge nested in a lipophilic benzimidzole structure, meaning the targeting of these dyes is dependent on mitochondrial membrane potential. In fixed cells, there is no mitochondrial membrane potential to start with, and the staining results are confounded. 
**
Lastly, some dyes have phototoxicity (they could either induce it directly or sensitize other molecules to become photoreactive and damaging). The deep red core is different from that of the red and green trackers. Therefore, there's no sure way to say that one dye's behavior will predict that of another. 
I recommend you reduce the deep red tracker dose and do the staining in live cells (followed by fixing if needed, and check if the the localization is retained). 
Good luck
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We noticed the increasing of  white blood cells and hemoglubin when patients with dengue fever give dexamethasone injection
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Thank you Dear Makarova
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what is the most important ROS and antioxidant that have important role in blood pressure reduction by exercise programs in hypertensive individuals?
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All the afore mentioned antioxidants have been shown to decrease systemic blood pressure in animal,but their efficacy in humans has not been proven. Mitochondrial oxidative stress seems to play a major role in the pathogenesis of both systemic and pulmonary hypertension. Recent studies have also shown that ROS produced by the mitochondria induce the activation of NADPH oxidases, leading to ROS induced ROS formation and oxidative stress.Targeting antioxidant directly to the mitochondria has been challenging and can produce harmful effects. Studies are ongoing to target mitochondrial oxidative stress.
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Is there any tester for oxidative (stress) status in humans?
Something as fast and easy-to-perform as a glucometer for glycaemia
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Measurement of oxidative stress involves direct as well as indirect methods. Many exciting new techniques are now available for measuring ROS directly like electron paramagnetic resonance, histochemical staining methods, Mass Spectroscopy, cytochemical methods, ROS tracer dyes, etc.  Non-invasive techniques using real time imaging of redox changes have been used in animal studies. Kits for estimating enzymes like SOD, GPx, etc are available. Estimating GGT may be a quick and easy to measure enzyme for oxidative stress.
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I need to perform accelerated stability calculations for cytochrome c and NADH. I have lyophilized samples stored at different temperatures for different time. How can I obtain the percent recovery of these samples for applying the  Arrhenius equation to calculate activation energy? Can i just calculate concentration using OD and use that to get percent recovery?
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I would do same enzyme activity assay for those samples to see the changes. 
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Hi everyone,
I am looking for a specific chemical inhibitor of MnSOD (SOD2). It should be able to pass the plasma membrane freely.
Available literature is not helping.
Thank You very much in advance.
Regards
Stefano Falone
University of L'Aquila - Italy
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I'll try to look some specific reference for your suggestion.
Thanks a lot
Stefano
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Hi,
I have to find a specific chemical activator of glyoxalase 1 (GLO1). It should have the capacity to pass the cell membrane roughly freely.
Thank You very much in advance.
Regards
Stefano Falone
University of L'Aquila - Italy
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thank you very much for your kind help
Regards, Stefano
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I compare infected cells and not infected cells, then I measure proteins of everybody even if I treat with antiobiotics. I cannot standardize with this method...
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If you know the number of cells and extract out entire SOD (along with other enzymes) from cells, obviously you can measure activity more precisely. Accordingly, you can divide over all activity (in terms of nanomoles or micromoles of superoxide anion radicals dismutated) with number of whatever cells you have taken.  
However, if you want to measure in vivo, theoretically Yes, but practically it may be impossible for following reasons: (1) Most importantly, how would you take care of supplying same quantity of superoxide anion radicals to both infected and control cells? Can superoxide anion radicals penetrate across the membranes?; (2) Would infection promote generation of superoxide anion radicals (generally any stress would enhance the generation of superoxide anion radicals)?
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I am trying to optimize the concentration of hydrogen peroxide concentration which can be used as positive control for ROS generation. am using DCFDA as molecular probe. I found that 100 uM H2O2 concentration is showing highest DCF fluorescence. I am using HeLa cell. Is the H2O2 concentration fine?
For my another experiment, I want to load DCFDA in to cell before any treatment and want to do my experiment for 24 h. I tried with DCFDA but it works only for 4 h. After that its signal is going down. Also I have to use serum containing media during my experiment. I saw in presence of serum, H2O2 treated cells also showing less DCF signal compared to untreated cell. So, I want some other probe which will work in presence of serum and should work for long time. Please give me suggestion. 
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The serum could be quenching some of the ROS and thus decreasing your DCF signal. You could try staining cells with DCF either for the last hour of your treatment or afterwards (if you can remove the treatment after the 24h). Or are you trying to measure ROS throughout the 24h treatment?
Amplex Red is another reagent that measures H2O2 production, but it is best for measuring ROS in the media (eg. if H2O2 diffuses out of the cell, or if extracellular superoxide dismutates into H2O2). As Jeff and David mentioned, DHE reacts mainly with superoxide. If you want to identify the precise species of ROS, certain methods are better than others (eg. DHE by HPLC versus flow cytometry, roGFP for ratiometric analysis using microscopy...). 
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I am getting different concentration values for the same sample. For liver tissue, at first I got 35 µM concentration. But, after one day the concentration value has come 13.5 µM.To assay MDA from tissue I have followed the protocol below-
  1. Homogenized in Tris-HCl (50 mM, pH-7.4).
  2. Centrifuged at 12,000g for 10 mins.
  3. Taken supernatant.
  4. 50 µl homogenate were mixed with 500 µl of 0.67% TBA, 500 µl 20% trichloroacetic acid.
  5. The mixtures were incubated in a boiling water bath for 20 min.
  6. After cooling to room temperature, the reaction mixture was centrifuged at 4000g for 10 min and the absorbance of the supernatant was measured at 532 nm.
For standard curved I have used 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM MDA dissolved in homogenizing buffer.
Every time I used to make standard curve prior to experiment. How should I troubleshoot my problem? Thanks in advance.
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Dear Debaprasad Koner, 
I would recall the same issues suggested by Pierre, the only difference that in our lab a 20% TCA and therefore TCA/TBA ratios varied from the mentioned  (~1/1) without problem. The issue with dissolving TBA deserves special attention, especially because it also changes the fluorescence levels depending on time and preparation procedure.  In addition to that: 
1- You could consider to add an antioxidant as BHT in your homogenates to prevent further MDA production during sample preparation.
2- You could add an emulsification solution as SDS to the assay to improve reaction rates. (If your samples contain relevant fat amounts I would also dilute them and measure by fluorescence instead).
3- You could consider a final extraction with a solvent as N-butanol to measure absorbance/ fluorescence only in the fraction really reactive of your assay.
Please, find attached a protocol which we have been using for a while with high reproductibility.
All the Best
Abel
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What evidence does support the suggestion that oxidative stress occurs first and then leading to mitochondrial/lysosomal dysfunction? I would be glad if you answer.thanks
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Dear Ali, I send the link to a publication that could serve as a basis for resolving questions.
Sincerely
Guillermo Schinella
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I wish to measure ROS production in fresh tissue homogenate (brain, liver, heart), but I am struggling to find a proper method to do that. AmplexRed does not seem ideal in this case because of the high peroxidase activity of tissue homogenate, so I was wondering if DCFH-DA could work properly? Any other suitable method will be appreciated too.
Thank you in advance for any advice!
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As stated by other ROS are shortlived and this is this short life (high reactivity) that explains their deleterious nature. Even H2O2 is short lived if catalase is present.
I find a little bit surprising to imagine that a "somehow measurable pool of ROS" in the homogenate would represent the oxidative stress status in the living matter rather than revealing possible interactions between the homogenate components and the oxygen of surrounding atmosphere. And this without taking into account the questionable nature of "ROS probes".
I acknowledge this is not "optimistic"! Then what I would suggest is to consider possible damages or molecules derived from ROS attack that would integrate the ROS attack in vivo. Possible parameters are:
- GSH/GSSG ratio
- Aconitase/fumarase ratio
- Oxyblot of proteins.
- modification of DNA bases, breaks...
- Oxidized lipids.
Then the history of the homogenate should be taken into account, for example in our experience the Aconitase/fumarase ratio is affected by freezing/thawing.
I hope it helps somehow.
FB
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I have some metal stress plants, and I want to analyse Hydrogen peroxide scavenging effects by the method of Ruch et al (1989).
I have 1 cm quartz cuvette. so please guide me how much H2O2, and Leaf extract i can used here?
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Kindly help with the protocol of end point assay for GSH and GSSG. Thanks in advance.
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More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.
 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.
 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.
This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1
More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.
 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.
 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.
 This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1
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Method for electrolyte leakage is widely used on plant sample to measure its response to stresses. Can the method also be used as one of the oxidative stress biomarkers for fish/shrimp sample?
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In my opinion there are other methods to measure Oxidative stress. Lipid markers are F2-Isoprostanes, MDA, Lipid hydroperoxides, 4-HNE. 
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Can any one give me an idea about oxidative stress panel that can be used on peripheral blood in vitro analysis of the possibility of oxidative damage for a drug??
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8-isoprostanes, 4HNE, MDA, protein carbonyls, nitrotyrosine, dityrosine. 
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We are following HR and NHEJ pathway. Want to know the assays and techniques to go ahead
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Dear JK,
The attached file is a recent published review that covers the answer to your question.
Effective Biomarkers and Radiation Treatment in Head and Neck Cancer
Thomas J. Ow, MD, MS; Casey E. Pitts, BA; Rafi Kabarriti, MD; Madhur K. Garg, MD, MB, BS
Context.—Radiation is a key arm in the multidisciplinary treatment of patients with head and neck squamous cell carcinoma. During the past 2 decades, significant
changes in the way radiation therapy is planned and delivered have improved efficacy and decreased toxicity. Refined approaches in the application of radiation and chemoradiation have led to organ-sparing treatment regimens for laryngeal and pharyngeal cancers and have improved local and regional control rates in the postoperative, adjuvant setting. The molecular and genetic determinants of tumor cell response to radiation have been studied, and several potential biomarkers are
emerging that could further improve application and efficacy of radiation treatment in head and neck squamous cell carcinoma.
Objective.—To discuss the current understanding of potential biomarkers related to radiation response in head and neck squamous cell carcinoma.
Data Sources.—Existing published literature. 
Conclusions.—Several potential biomarkers are actively being studied as predictors and targets to improve the use and efficacy of radiation therapy to treat head and neck squamous cell carcinoma. Several promising candidates have been defined, and new markers are on the horizon.
(Arch Pathol Lab Med. 2015;139:1379–1388; doi:
10.5858/arpa.2014-0574-RA)
CONCLUSION
This review has highlighted several biomarkers that are actively being studied as predictors and targets to improve the use and efficacy of radiation therapy to treat HNSCC. Several promising candidates have been defined, and new markers are surely on the horizon. Biomarkers that lead to significant improvements in patient outcome will require rigorous validation and prospective testing.
Hoping this will be helpful,
Rafik
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In erythrocytes gamma glutamyl cysteine ligase represents the most important enzyme involved in GSH synthesis.In fact, a lot of drugs or compounds can modulate its enzyme activity either during oxidative stress or no stress condition. 
A simple method to purify and evaluate its activity could be suitable.
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Dear Arbace,
gGLYCys activity determination in red cells is difficult becouse it is very low. So if you must measure it by spectrophotometry it is a problem. In fact Hb which adsorbs at 340 make impossible this determination in this kind of biological matrix becouse you have to use Hb separation procedures and/or enzyme purification.
On my opinion you should follow this procedure but it requires HPLC separation of the products and fluorescent detection. However even if it is time consuming it can be applied in the presence of Hb requiring only the absence of relevant gGT activity (which is exactly the case of red cells by the way). We use this method.
C.C. Yan, R.J. Huxtable, J. Chromatogr, B. Biomed, J. Chromatogr. B Biomed.
Appl. 672 (1995) 217–224.
Briefly, a solution containing 10 mM ATP,
150 mM KCl, 2 mM K3EDTA, 20 mM MgCl2, 10 mM glutamic acid,
2.5 mM cysteine, 0.5 mM DTT in 1 M Tris/Cl, pH 8.1, was let to
incubate at 37 C for 15 min. Thereafter, 10 ll-aliquots of sample
were added to 150 ll of the incubation solution; after 5 min at
37 C, the sample was deproteinized by 1:1 (v/v) addition of
12% (w/v) TCA containing 1 mM K3EDTA. 0.1 ml of the obtained
supernatant was then reacted with 2.5 mM (final concentration)
mBrB in the presence of 2 M Tris (25 ll). After 10 min of incubation
in the dark at room temperature, samples were acidified by
addition of 5 ll of 37% (v/v) HCl and analyzed by HPLC for the
thiol content as above described.
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Dear all:
Anyone know about the effect of storing time at cold temperature (-40, -80ºC) on several biomarkers of oxidative stress, on blood?
Any comment would be highly appreciated.
Many thanks and best regards,
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Dear Chibuike C Udenigwe:
Many thanks for your answer!
Could you be so kind to give me an example of conditions that can lead to further oxidation?
Best regards,
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Hi, I was wondering if anyone has used H2O2 added externally to adherent cell cultures as a positive control to measure ROS production. What is the logic behind this (are you not just measuring the H2O2 that you've added)? How much H2O2 do you add? Do have a good protocol for performing this experiment?
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I agree with Dr. Vidal and Dr. Mukherjee. It's better to induce endogenous ROS, rather than using an exogenous ROS to augment them, because species like H2O2 have broad and nonspecific effects if applied globally. Even GSH will be reacted upon. It's hard to say what's being mimicked. 
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 I want procedures for catalse , super oxidedis mutase , para oxinase enzymes for blood sampels???
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Your question involves three different enzymes, so the discussions and answers might not be easy to follow. I would suggest you to ask a different question for each enzyme, next time.
Below are some references for catalase and superoxide dismutase, with detailed protocols. I could not find a PDF for the oldest references, but the papers with more recent modifications of the protocols might be more useful anyway.
As for para-oxydase, were you referring to the PON1 enzyme? I attached a few papers on that as well.
I also link to other threads in researchgate with similar questions.
All the best with your work!
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I am looking for any biologically relevant oxidants other than H2O2....
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Peroxynitrite, superoxide could be very relevant. Myeloperoxidase could be very good source of HOCl in vitro.
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I wonder  half life of OGG1.I have never found to anywhere.Is there differentness between wild type OGG1 and OGG1-S326C in terms of half life? Thanks in advance for your interest. 
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Under normal conditions the OGG1 protein half life is much longer that what is reported in the paper cited by Christian. As far as I can judje from our own RNAi experiments, OGG1 half-life in HeLa cells must be >10 hours. We checked the protein expression by Westen blot (normalised by actin). One needs to wait 40-48 hours to obtain a significant knockdown.
To your second question: there is no reason why the lifetime should differ between the 326 Ser and Cys variants, at least under normal conditions. However, under stress this might be different. Some believe that the Cys variant is more prone to oxidation, but I do not know any data showing that that would promote protein degradation.
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Can anyone suggest some good procedures to measure hydrogen peroxide inside living cells.
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Dear Dr. Scheckhuber,
Thank you very much for your suggestions.
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I want to know the assay for DNA damage in response to pathogen and heat stress and also due to reactive oxygen species produced in stress response
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Hi,
Comet assay detects single strand breaks, please see my profile to get all the information that you need.
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I've been digging through piles and piles of literature for studies of ROS mediated control of signaling cascades - does anyone have any good article suggestions?   (feel free to toot your own horn here!)
There is a surprisingly large amount of poor or confusing info out there - I'm trying to speed my search and not get distracted by misleading studies 
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Hi Levi,
I'm not sure what the basis of your search is of course, but if it is from a molecular point of view these reviews might be interesting to you: 
This review by Claudia Cremers and Ursula Jacob is very informative: http://www.jbc.org/content/early/2013/07/16/jbc.R113.462929
If you're new to redox signalling and biochemistry, this perspective might be very useful to you as well (it might help to interpret many of the data out there): http://www.ncbi.nlm.nih.gov/pubmed/21459321
Also, we have recently written a review on disulphide-dependent signalling that includes some examples of (disulphide-mediated) redox signalling events that might be of interest: http://www.ncbi.nlm.nih.gov/pubmed/25109988 
Finally, this is quite a long one but there might be some leads in there for you: http://www.ncbi.nlm.nih.gov/pubmed/24634836
Best,
Marrit
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oxidant and antioxidant parameters other than catalase, glutathione, glutathione peroxidase, glutathione reductase, total thiol, glutathione -S- transferase and total antioxidant capacity as antioxidant.
in addition, malondialdehyde (MDA), lipid hydroperoxide (LOOH), conjugated dienes (CD) and nitric oxide as oxidant.
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Not new but superoxide dismutase activity can be utilized as an antioxidant parameter.
What do you want to do? Why do you need new oxidant and antioxidant parameters? 
Cheers, Christian
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I would like to measure resistance to oxidative stress and relate it to different levels of methabolic activity. What are the best markers to measure in these circunstances, among the wide spectum of possibilities?
Many thanks!
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I think it is the singlet oxygen or super-oxide molecules that are produced due to single electron reduction and consequently leaking of electron from complex III of mitochondrial electron transport chain. A little amount can be produced from complex I also. Production of superoxide is a direct consequence of transferring electrons from one complex to another complex in mitochondrial electron transport chain.
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Hi guys, I am recently testing a drug that is supposed to increase the superoxide production in cancer cells. However, I noticed the DHE staining (cytosolic superoxide) increased whereas the mitoSOX (mitochondrial superoxide) decreased. Any suggestion/explanation for this? I think it's worth trying the staining of the mitochondrial membrane potential in parallel with mitoSOX because as I know the Mitosox Red accumulates in the mitochondria on the basis of its membrane potential.
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HE is not really cytosolic, because of its positive charge, marking it easy to penetrate membrane and prone to bind DNA. Prolonged incubation of HE results eventually in a heightened background of fluorescence that is not contributed by specific reaction with superoxide. Therefore overincubation should be avoided. HE preferentially localizes to nucleus in the end points. 
MitoSOX has been recommended by Invitrogen to be used at doses between 1-5 uM. At 5uM, for over 30 min, the probe becomes visiaully toxic to some cells, and triggers off more ROS production, artificially. 
In imaging, MitoSOX also contributes to some (nuclear) DNA staining if incubated for 30 min or longer. 
In theory, an ROS probe can work like a scavenger towards some ROS, esp. when its probe concentration is relatively low to yield a detection fluorescence (e.g. below 1 uM).
MitoSOX is recommended to be used at about 2-.2.5 uM, 15 min to 20 min. 
Mitochondria have their own DNA, so specificity of MitoSOX has been in debate. 
See: 
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This question is with regards to free metal ions and oxidative stress.
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Transition metal ions in their free state bring unwanted biological oxidation's generating  reactive oxygen species and hence oxidative stress. The Coordination of such free metals in body with certain bio-ligands can modify the redox potential of these free metal ions which can block their redox reactions leading to the production of ROS. This methodology can be indispensable in prevention of metal based oxidative stress by blocking the redundant bio-redox reactions.
a ref can be
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Buffers such as Krebs-Henseleit require continuous gassing with 5% CO2 to maintain a pH of 7.4
In a larger reservoir this is fairly easy to do with simple aquarium equipment such as an airstone or a diffuser. However, these options are (in my experience) less viable in smaller tissue baths; the stones are simply too big.
How do you maintain proper oxygenation in small baths?
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Bubbles could be not good for tissues and isolated organs.
As option, oxygenate your solution in other reservoir and recirculate solution via membrane filter to your small bath.
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Does anyone have experience with the CellROX Green reagent for oxidative stress detection? We are trying to detect oxidative stress in cells(live or fixed) in 35mm plastic dish.
Unexpectedly, the cells treated with ROX reagent gradually become to express strong signals while we observe the cells using fluorescence microscope. This unknown strong signals are detected in all cells which are exposed to excitation ray.
H2O2 treatment is done for 4 hours. The last 30 minutes was incubated by adding Cell ROX Green(5uM).
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I notice that your sample images contain fixed cells. Under this condition, there's no way for fixed cells to produce ROS (notably superoxide, then H2O2) in logically predictable ways.
It's more likely the fluorescence increase is due to auto-oxidation of the probe, in the presence of laser photon. In other words, photo-activated auto-oxidation. 
This makes the probe not to be called robust enough for imaging. 
****
Our lab has just published a JACS paper on superoxide detection. 
You may consult your supervisor, whether it's desirable to write and request the probe from our lab. We distributed a requested aliquot to a US lab, just one week after the paper was published. 
However, the decisions of whether to grant the request rest on my boss' discretion.... :)
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I need to know, does anesthesia using light halothane inhaling in rats affect the measurement of neurotransmitter levels and the measurement of oxidative stress parameters in the brain or not?
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As light halothane anaesthesia lowers sympathetic tone, you will measure lower levels of plasma catecholamines.
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How can we explain the significant decrease in GST activity, while there is no oxidative stress (significant decrease in MDA levels) and the GSH levels are normal? Also, SOD levels showed significant increases, while catalase activity showed significant decrease.
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GST as an enzyme has many detoxifying functions not related to oxidative stress, so it can change completely independently.
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I want to study the effects of aflatoxins on the brain in terms of oxidative stress
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Dear Musa
Important biomarkers of oxidative stress in the brain are lipid peroxidation, protein carbonylation and nitrosylation, DNA and RNA oxidation and the ratio between reduced and oxidized glutathione (GSH/GSSG).
Lipid peroxidation markers widely used include 4-hydroxy-2,3-nonenal (4-HNE), acrolein, F2‑isoprostanes and thiobarbituric acid-reactive substances (TBARs; such as malondialdehyde (MDA)). These are really useful since the brain is enriched in polyunsaturated fatty acids (PUFAs) that are highly susceptible to peroxidation.
Protein oxidation markers include protein carbonyls and 3-nitrotyrosine.
8‑hydroxyguanosine (8OHG) and 8‑hydroxy-2-deoxyguanosine (8‑OHdG) are markers of RNA and DNA oxidation.
These products are used as markers of oxidative stress in human brain and you can find several papers showing their presence in human postmortem brain tissue.
Since endogenous antioxidants and antioxidant enzymes are frequently increased as a mechanism of protection against the oxidative insult they are also used as markers for the occurrence of oxidative stress. 
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This will be done in renal disease patients.
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We have used NBT from Sigma, Himedia, Merck, SD-Fine chemicals etc. NBT from all these sources worked equally well.
As far as NBT assay is concerned for measuring oxidative stress is concerned, I have reservations. NBT can be reduced by number of biological events, almost all dehydrogenases can reduce Tetrazolium. I am attaching a good publication, wherein researchers reported that NBT can even generate superoxide anion radicals (many of us use it for measuring superoxide anion radicals).
It is not clear, what exactly you want to measure (what aspect of oxidative stress)? Are you interested in measuring total antioxidant capacity or lipid peroxidation or levels of enzymatic & non-enzymatic antioxidants?
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Hi,
I am trying to measure the content of methylglyoxal-dependent damage on arginine residues in my protein samples. I know an extensive protein hydrolysis must be carried out in order to detect the MG-H1 adduct, however the standard procedure (24-h incubation with 6N HCl at 110 °C) reduces the yield of the adduct recovery. Alternatively, longer incubations can be carried out for several days in presence of several proteases (pepsin, thymol, pronase E, aminopeptidase, prolidase). Can anyone suggest me which products to use and how to proceed exactly, please? Thank You very much in advance.
Stefano Falone, Ph.D.
Dept. of Life, Health and Environmental Sciences
University of L'Aquila - Italy
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Dear Leonardo,
thanks so much for Your kind reply. I'll try. I'll contact You if further help is required.
Happy Easter!
Stefano Falone
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Is it preferable to combine them?
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Dear Joshua
It depends on the question you need to answer. For example for the patients that I follow, protein based biomarkers (or sometimes functional assays looking for the functionality of these proteins) are fundamental for the phenotypic diagnosis of a disease, and genotypic markers are important for genetic counseling.
Moreover, some genes present mutations affecting different domains of the related protein causing astonishing different phenotypes (e.g.: STAT1 loss of function mutations, that can be recessive or dominant with susceptibility to different pathogens, and gain of function mutations with other different phenotypes).
So, for some diseases the genotypic-phenotypic correlations are not so direct and both assays are important to understand the biological phenomena.
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I need a kit that is sensitive and not time consuming. Thank you.
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 Hi
I test this assay the results very good
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The mitochondria is the primary target for oxidative stress induced by Cisplatin, resulting in loss of mitochondrial protein sulfhydryl group. We have some interesting data on Oxidative stress biomarkers - CAT, SOD and GPX. But, I need some help in estimating the mitochondrial protein level for better conclusion. ANy suggestions please ?
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Hi Shaloam,
MitoSciences offers several kits to assess mitochondrial abundance and activity.  This kit: http://www.abcam.com/Complex-I-Human-Protein-Quantity-Dipstick-Assay-Kit-ab109722.html will allow you to quantify mitochondrial complex I, which can be an appropriate proxy for total mitochondrial protein content.  You can browse around their website for other options too.
Alternatively, you can perform a Western blot for TOM20 or other mitochondrial markers as a proxy for mitochondrial protein abundance.
If you have a fluorescence microscope and analysis software that can quantify fluorescence signals, you can also stain your cells with MitoTracker Green and quantify the signal as a measurement of total mitochondria.
I have used each of these in the past with good success.  All of these approaches are pretty straight forward and routinely accepted in the literature as indicators of mitochondrial abundance. 
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I want to measure monkey neopterin according to GLP.
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I would suggest to reach out to Prof. Dietmar Fuchs (dietmar.fuchs@i-med.ac.at) from Innsbruck Medical University, Austria. He is one of the world's experts on neopterin and has pioneered the development of several assays to measure neopterin. I am sure he will be very helpful. Good luck!
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I want to measure oxidative stress in intestine by using H2DCFDA;. How can i prepare my tissue?
sINCERELY
sABRIA
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Thank you very much for you answers
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In order to analyze long-term changes of oxidative stress, would it be sufficient to measure urine F2 isoprostanes in a single urine sample in humans (i.e. morning sample) or should it be measured in 24h urine?
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I agree with Pascale. Quantification of isoprostanes intended to reflect their production for a long period, representing production chronically. Therefore, it is convenient measurement in 24-hour urine stored at 4 ° C / if possible) and supplemented with BHT to prevent oxidation of the molecules of interest. Regarding the measurement method, I also agree with Pascale is preferred GC / MS, with all its drawbacks for HOMEWORK and derivatization of samples, but is that Elisa available, in my opinion and experience, do not offer nor good practicability and good accuracy of results
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The hypothesis of James Watson the co-author of the DNA double helix about the loss of ROS generation and it's role to generate diseases as type II diabetes and cancer, was criticed by a part of scientific comminity and supported by the other part, could you give some papers or more details about the two point of view, Thanks in advence,
You find attached the hypothesis paper.
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i'm now preparing a minireview about this sbject and i'll publish it soon, anyway it's a good idea need to be more and deeply investigated
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Are there any bio-markers we can check in addition to malondialdehyde in order to measure the extent of lipid peroxidation?
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8-isoprostane is supposed to be a good marker of phospfolipid peroxidation.
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I have fully reduced and oxidised bovine serum albumin to make protein carbonyl standards. I just ran a BCA assay to determine the protein concentrations of both and discovered that the reduced protein concentration is 9mg/ml and the oxidised is 6mg/ml. What is the best way to adjust the reduced protein concentration so it's the same as the oxidised solution?
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Hi Matthew
do you have a protocol for generating oxidized BSA that you could share with me?
cheers
dave
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is the increasing level of CAT SOD GPx GPOx activities go with GSH high level?
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There is no clear relationship between GSH levels  and the realm of 'antioxidant' enzymes. GSH biosynthesis is under the control of the NRF2 system, as are some of the peroxidases, not all. For sure, the 8 GPxs, the 6 peroxiredoxins and the 3 SODs are not regulated the same way. Read the relevant literature before starting meaningless measurements (small collection attached).
Best regards
Leopold Flohé
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Free radicals and mitochondrial dysfunction are proportionally correlated.
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Yes there is a correlation. Use ETC inhibitors and check the modulation in ROS using mitosox red. Also simultaneously monitor the changes in antioxidant levels. You will definitely get your answer.
Regards
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My experiment was assigned to eight different groups 
I want to compare the mean concentration/activity of oxidative stress markers ..(CATALASE, GPx,GSH, TBARS.......) between those groups AFTER treatment period. 
Is it acceptable if I use One-Way ANOVA  or must I use Two-Way ANOVA ?
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Unless you have a hypothesis that applies to all 8 groups, a 2-way is appropriate.  That is, if you have a hypothesis that group 1 will have a positive effect, but groups 2-7 do not, then you can test with one-tail.  However, you may be missing out on unexpected differences if you don't use two-tail.
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Several authors in the past have reported TBARS as lipid peroxides or simply as malondialdehyde (MDA). In reality it may not be the exclusive end-product of free radical (FR) activity. Circulating levels of these compounds may not truly reflect FR activity
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TBARS are thiobarbituric acid( TBA) reactive substances. They are different types of aldehydes. due to the presence of reactive oxygen species in the cells lipid peroxidation occurs. Intermediates of lipid peroxidation will ultimately converted to different types of aldehydes ex formaldehydes, acetaldehydes etc. Malondialdehyde is the end product. So malondialdehyde is the commonly accepted biomarker of lipid peroxidation TBA is the reagent used to determine the amount of aldehydes or the products of lipid peroxidation. All types of aldehydes are reactive with TBA. It is aldol condensation reaction. After the reaction with different aldehydes with TBA, TBA-aldehyde adducts gives different coloured compounds. It is not an free radical assay it is a test for estimation of lipid peroxidation with its end products you can check out my publication: Ultra sensitive detection of malondialdehyde with surface enhanced Raman spectroscopy.http://download.springer.com/static/pdf/566/art%253A10.1007%252Fs00216-010-4225-3.pdf?auth66=1406078746_ae8cf38aeae9ec1b11c9493e35db32d6&ext=.pdf
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Actually I got some papers, are they showing same results in blood?
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A sensitive measurement of total body antioxidant level would be reflected inside of the cell. In my opinion, if you were to get a more meaningful measurement of glutathione, it would be looking at lymphocytic glutathione levels, GSH/GSSG.
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Howitz and Sinclair state in a brilliant manner: “Our xenohormesis hypothesis proposes that animals and fungi (heterotrophs) have evolved the ability to sense signaling and stress-induced molecules from other species, and that they are under selective pressure to do so. In essence, xenohormesis refers to inter-species hormesis, such that an animal or fungal species uses chemical cues from other species about the status of its environment or food supply to mount a preemptive defense response that increases its chances of survival” Can anybody provide examples in which this hypothesis is unlikely?
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Agree with Artur!
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