• Tatiana Y. Karogodina added an answer:
    Anyone has experience on EPR on total tissue to measure superoxides and hydroxyl free radicals ?

    Hi, I am trying to measure superoxide levels using EPR with DMPO as a spin trap. I had tried in isolated mitochondria and I got nice spectra (only in presence of inhibitors). Now I like to use total tissue, kidney and heart. If any of you have tried this can you provide conditions such as buffers and  how you homogenize the tissue.

    Also, anyone has experience in injecting spin trap into animals  and measuring EPR later ? Ideally I want to perform in vivo measurements, or something close to that.

    Thank you 

    Tatiana Y. Karogodina · Russian Academy of Sciences

    Dear Manjula

    I suggest to see papers of Komarov DA. For example this one:


  • P. Pardha-Saradhi added an answer:
    What si the best protocol for NBT staining in tomato leaves for detection of superoxide radicals?
    Can anyone please suggest the best protocol for NBT staining (for suproxide radicals) in tomato leaves. Could you also suggest how, after staining, I can efficiently remove chlorophyll without damaging leaves and heat burning.
    P. Pardha-Saradhi · University of Delhi

    Dear Praveen,

    Following NBT staining for 12 to 24 h), you can remove all pigments (including chlorophylls) with the exception of formazon by dipping leaves in clearing reagent (3:1 ratio of TCA and phenol). Kindly handle clearing agent carefull taking all necessary precautions. You can leave leaves for 6 to 24 h depending on intensity of various pigments (if you want to hasten the process you may keep it in oven for 30 to 60 min at 60oC). Subsequently, decant clearing reagent carefully without damaging leaves (or you can lift the leaves carefully with a good blunt forceps and transfer to a fresh petriplate or a plastic box and add lactophenol. If you wish you can replace lactophenol after few hours. You can infact store your cleared leaves with nice colour of formazon in lactophenol as long as you want. Be careful in using clearing reagent. If you take proper precautions you will find this method to be most simple.

    Good Luck

  • Can anyone suggest specific scavenger(s) for superoxide but not for ROS?

    I am looking for the reagent which can scavenge superoxide in living cells. Most of ROS scavenger, like tempol, inhibit not only superoxide accumulation but also reactive nitrogen spices (RNS). But I am actually looking at the effect of RNS in oxidative stress but not superoxide. Dose anyone know the cell permeable superoxide scavenger?

    Francisco J Sánchez-Gómez · Spanish National Research Council

    We have used PEG-SOD, a cell permeable analogue to dismutate superoxide, but, you have to take in account the kinetics of the enzymes, doses and toxicity. Other compound you can use is TIRON. It is very effective and specific for superoxide.

    Free Radic Biol Med. 2014 Jun;71:146-56. doi: 10.1016/j.freeradbiomed.2014.03.011. Epub 2014 Mar 15.
    Acute hypoxia produces a superoxide burst in cells.

    Supplementary material.

  • Benson Solomon added an answer:
    Is there any other superoxide radical scavengers except TBA? Can you also give the referance?

    I want to identity that if the superoxide radicals were involved in Fenton reaction. I need to use superoxide radical scavengers except TBA. Thank you.

    Benson Solomon · University of South Carolina

    MCLA and lucigenin are two superoxide probes our group has used for superoxide measurements

  • Mathieu Pierre added an answer:
    What is best chemical to induce oxidative stress selectively by superoxide anions?
    But not by other ions such as hydroxyl etc. I wants to elevate superoxide level selectively in the cells.
    Mathieu Pierre · Université de Poitiers

    you can use DCMU

  • Praveen Roylawar asked a question:
    How to: superoxide radical detection by spectrofluorometer?

    I did Nbt staining in tomato leaves to detect superoxide radicals (Blue formazol formation). Now I want to detect the same radicals using a spectrofluorometer. Can anyone please explain to me how to do it ? Can I use the same sample (formazol precipitated sample) for spectrofluorometric analysis or do I have to use an unstained sample?

    Thank you.

  • Katarina Venglovska added an answer:
    Does anyone have any suggestions on doing an SOD assay using the Marklund and Marklund protocol?
    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.
  • Shane Vontelin van Breda added an answer:
    Can anyone give advice on Fenton reaction inhibition in M. tuberculosis?
    I am investigating the mechanism of action of a cationic peptide against MTB. I suspect one of the mechanisms is by the Fenton reaction. One way I would like to prove this is by use of thiourea and dipyridyl, as they have been used before in inhibiting the Fenton reaction.

    Does anybody have an idea of what concentrations I should begin looking at against MTB?

    Has anybody got any experience investigating and inhibiting Fenton reaction again against MTB?

    I would also like to ask about the preparation of thiourea and dipyridyl. Should it be added to bacterial media, then autoclaved? Or should the compounds be added to the autoclaved media using filter sterilization?

    Shane Vontelin van Breda · University of Pretoria

    Thank you Boris.

  • Rajesh Kumar Tewari added an answer:
    Why should blank samples not have PMS in scavenging activity of anion superoxide?
    I´m following this protocol:

    Superoxide radicals are generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of NBT. In these experiments, the superoxide radicals were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 1 ml of NBT (50 mM) solution, 1 ml NADH (78 mM) solution and sample solution of fraction (25 – 500 mg/ml) in ethanol. The reaction started by adding 1 ml of PMS solution (10 mM) to the mixture. The reaction mixture was incubated at 25 °C for 5 min, the absorbance was read at 560 nm by spectrophotometer (Schimadzu UV-Vis 1700) against blank samples using ascorbic acid as a control. Decreased absorbance of the reaction mixture indicated the increasing of superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the following formula:
    % inhibition = [(A0-A1)/A0] x 100
    where A0 was the absorbance of the control , and A1 was the absorbance in the presence of fraction or standards.
    Rajesh Kumar Tewari · University of Vienna

    Ascorbic acid can directly reduce NBT to formazan. I really do not see the relevance of using Ascorbic acid in your experimental system. 

  • Just Genius added an answer:
    Has anyone detected superoxide anions with luminescence method?
    For measurement of superoxide anions induced by Angiotensin II in VSMC cell lines, I used luminol and enhancer incubated with pretreated cells but the value of light intensity was only about 200 per 105 cells after 30 mins incubation. Could that be right? Why is the light intensity so low?
    Just Genius · AbbVie
    Hi again! As far as I know, luminol will primarily detect hydrogen peroxide, not superoxide. Lucigenin may be the better probe, but you may also consider fluorescent dyes (e.g. available from Molecular Probes). In the experimental setup you describe, light emission will be dependent on the presence of (intrinsic) SOD which converts superoxide into H2O2. The readings of your instrument are arbitrary and depend on the detector setup and the instrument settings (integration time, blank value substraction detector gain etc), so 200 counts may be entirely OK. You may use xanthine+xanthineoxidase as a positive control (it will generate superoxide anions). Or you treat some leucocytes with LPS and measure the oxidative burst (works even with full blood). This would be a nice "biological" positive control.
    Good luck!
  • Olivier Coste asked a question:
    Can antioxidant molecules interfere with an NBT assay for oxidative burst and give a "false negative"?
    If my molecule activates macrohpages oxidative burst but at the same time scavenges the superoxide radicals, is it going to give a false negative?
  • Reena Arora asked a question:
    Does anyone have experience with MitoSOX assay?
    I was wondering if anyone has done a MitoSOX assay in 96 well plate?
  • Devendra Kumar added an answer:
    When is the exact time that superoxide radicals are produce in plants?
    In a walnut.
    Devendra Kumar · Indian Agricultural Research Institute
    its also depend on type of pathogen, resistance gene...but most case within 30 minute........reference-NO way back: nitric oxide and programmed cell death in
    Arabidopsis thalianasuspension cultures
    Andrew Clarke, Radhika Desikan, Roger D. Hurst, John T. Hancock and Steven J. Neill*
  • Rajbardhan Mishra added an answer:
    What is the protocol for Reactive oxygen species(ROS) assay especially superoxide when we co-culture, neutrophils with cancer cells.
    Rajbardhan Mishra · Institute Animal Physiology and Genetics AS CR, v.v.i.
    Thank you so much. I hope it will help me.
  • Marcin Nowicki added an answer:
    Does anyone know simple protocols for analysase, superoxide, dismutase and ascorbate peroxidase enzyme activity in plants?
    The papers do not mention standard concentrations to be used for measuring enzyme activity, could someone help me with that?
    Marcin Nowicki · Research Institute of Horticulture in Skierniewice
    Nature Protocls has it all. I highly recommend it (successfully used an Ascorbate microtiterplate assay few years back).

About Superoxides

Highly reactive compounds produced when oxygen is reduced by a single electron. In biological systems, they may be generated during the normal catalytic function of a number of enzymes and during the oxidation of hemoglobin to METHEMOGLOBIN. In living organisms, SUPEROXIDE DISMUTASE protects the cell from the deleterious effects of superoxides.

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