• Yurii V Geletii added an answer:
    Which kind of light we should use in superoxide scavenging activity to induce the reaction?

    This is the protocol we used and we did not obtain any results: Superoxide radical was generated in the PMS-NADH system containing 3 mL Tris–HCl buffer (16 mM, pH 8.0), 338 M NADH, 72 M NBT, 30 M PMS and varying concentrations of ascophyllan fractions (10–50 g/mL). The mixture prepared earlier was incubated at room temperature for 5 min and the absorbance was read at 560 nm against the blank. Inthe control, the sample was substituted with Tris–HCl buffer. Thank you for help

    Yurii V Geletii

    Your question does not make any sense. 

    Correct the units in your description. "the PMS-NADH system containing 3 mL Tris–HCl buffer (16 mM, pH 8.0), 338 M NADH, 72 M NBT, 30 M PMS and varying concentrations of ascophyllan fractions (10–50 g/mL)" 

    Be professional. 

  • Biqing Bao added an answer:
    How to calculate the concentration of superoxide?

    I want to calculate the concentration of O2•− generated by enzymatic reaction of xanthine (X; 300 μM) and xanthine oxidase (XO; 0.01 U/mL) in  potassium
    phosphate buffer (0.1 M, pH 7.4), i am curious how to detect exactly the concentration of this active species?

    Biqing Bao

    Thank you both for the valuable suggestions! However, I still have some questions. According to the references( such as this AC paper | Anal. Chem. 2014, 86, 7071−7078),the superoxide was generated by KO2 in DMSO and was determined by the reduction of ferricytochrome c in water using the extinction coefficient (21.1 mM−1 cm−1) of ferrocytochrome c at 550 nm. My question is that: (1) the superoxide disappeared quickly(second order) in the aqueous solution, does the quantity of superoxide calculated from this method is much lower than its actual quantity?(2) extinction coefficient of ferrocytochrome c at 550 nm in different papers is quite different, why?

  • Karel Chalupsky 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 

    Karel Chalupsky

    I recommend to use DHE with HPLC detection of oxyethidium (adduct of superoxide and dihyroxyethidium) as independent control of EPR experiments. Fe ions with tissue lysates are major problem they can oxidase spin labels at least in my experience.

  • Lucy Evans added an answer:
    Does anyone have a ROS detection protocol for flow cytometry using the ROS/superoxide kit?

    Hello Everyone,

    I want to detect ROS production in the cells am using. I therefore bought an Abcam (ab139476) detection kit. 

    There is a statement on the protocol I do not understand..

    "If the vehicle, experiment agent and ROS inducer will be added to the ROS/Superoxide Detection solution (2X), their concentration need to be 2X. For the negative control, 1X of the inhibitor needs to be compensated in the solution to maintain its final concentration at the same level."

    1. Does it mean that if I add 100microliter of ROS/Superoxide detection solution, I need to add 100 microliter of treatment, vehicle or inducer?

    2. For the inhibitor, does it mean I have to aspirate the previous solution and add a fresh one together with the ROS detection solution?

    Also in this statement,,,,,,

    "To every 10ml of 1X washing buffer or culture medium, add 4microliter Oxidative stress detection reagent (Green) and 4 microliter superoxide detection reagent (orange).

    Gently mix to make 1:2500 dilution staining solution. To prepare smaller volumes of 2X ROS/Superoxide detection mix, intermediate 1:10 dilution of both green and orange detection reagents in 1X Wash Buffer or culture media is recommended."

    3. What does this exactly mean? if you can use an example that would be really helpful, Thanks.

    4. If you have used a similar (don't have to be the same) kit, could you please be kind enough to share the protocol, thank you.

    Lucy Evans

    Thanks guyz, you suggestions were really helpful. I did the experiment and it was a success.

  • Khalil Rajeh added an answer:
    Please anybody can help me, what is the underlying mechanism of hydrogen peroxide to increase ROS&RNS in Huvecs?


    ONOO –

    OH −

    Khalil Rajeh

    Thanks all, Dear David C. Ellinsworth, H2o2 can also deactivate Akt as well as loss of  eNOS Ser1177/1179 phosphorylation in long term treated cells.

  • Brian M Polster added an answer:
    MitoSOX issues in fibroblast cell lines

    I am trying to assess mitochondrial superoxide in three different cell types but I am experiencing some issues - for two of these cell lines the staining works well and I can see that the dye has localised to the mitochondrial network, however, within the third cell line the staining appears diffuse - I can see mitochondrial networks at the edges of the blurry stain but in the middle it is just a red haze.

    I have done mitotracker green analysis to look at mitochondrial content however the difference in mitochondrial content was not so large that I would expect the MitoSOX signal within this cell line to not produce networks due to too much signal. In addition, the intensity of mitoSOX is not always greater (sometimes equivalent) to that in the other cell lines. If anyone has any suggestions or has had a similar problem I would be really grateful for any suggestions.


    Brian M Polster

    MitoSOX is a very difficult dye to work with; it should be used cautiously and supported by other techniques.  It needs to be titrated for every cell type and concentrations much lower than those recommended by the manufacturer (e.g. 0.2-1.0 microM rather than 5 microM) are usually optimal.  See and

    Best of luck, Brian

  • Bensouici Chawki asked a question:
    Is there a good protocol to superoxide anion scavenging activity on 96-well microplate reader?

    Superoxide anion scavenging activity

  • Isacco Gualandi added an answer:
    How can the concentration of peroxyl radical be determined in seawater?

    Free radical are photochemically generated in seawater such as hydroxyl radicals, nitric oxide radical, superoxide radical and probably peroxyl radicals. Is there any developed method for its measurement?

    Isacco Gualandi

    The very high reactivity of such radicals is responsible for their  short half-lifes (about 10−10 s for hydroxyl radical) which explains why their concentration is generally very low. Their direct determination is quite difficult for some matrices. For example the in vivo measurement of OH radical by electron paramagnetic resonance (EPR) fails because the presence of scavengers leads to an extremely low steady-state concentration. The simplest way to detect them is indirect, i.e. by using a probe which reacts very quickly with them. In function of the probe that is used, different detection techniques can be used.  Nevertheless such kind of determination usually estimates the total amount of produced radicals and not their concentration (I Gualandi, D Tonelli Talanta 115, 779-786, 2013; C. von Sonntag Free-Radical-Induced DNA Damage and Its RepairSpringer, Heildenberg (2006)).

    I give you some additional information.

    Determination of hydroxyl radical is ussualy performed with the use of a probe. The product of the reaction between the probe and the radical are determined by HPLC. Several probes are used. The most common is the salicylic acid but the accuracy of the measure is debated. This paper can interest you (Determination of hydroxyl radicals photochemically generated in surface waters under sunlight by high performance liquid chromatography with fluorescence detection, Anal. Methods, 2014, 6, 8193).

    Superoxide radical can be determined also without the use of a probe. Some electrochemical sensors exploit the superoxide dismutase to reach a good selectivity. Also the probe can be used.

    The determination of peroxyl radicals is quite difficult. I carefully searched some papers that described their determination for a different matrix (atmosphere) some years ago, but I found nothing.  

  • Pushpendra Singh added an answer:
    Can anyone suggest simple calorimetry based assay for detection of Reactive nitrogen species (RNS) and superoxide?

    Actually on the stress condition cells produce ROS and RNS that ultimately leads to cellular oxidative stress. HOW can detect RNS and superoxides

    Pushpendra Singh

    Thanks dear, Nai-Kei Wong  sir

  • Ricardo Pérez-Pastén added an answer:
    What's the best procedure to determine SOD activity in trout ?

    I want to determine SOD activity in liver, gill and muscle and in many publication the method use "xanthine method" or "pyrogallol method"

    Ricardo Pérez-Pastén

    We used RANSOD SOD kit from Randox Company. We adapted to 96 well microplate and results were consistent and reproducible. With this setting, the kit gave us 80  tests per 20 ml reagent  bottle.

  • Radia Slimani added an answer:
    Is there any simple and reliable superoxide anion scavenging assay for testing antioxidant?

    Is there any simple and reliable superoxide anion scavenging assay for testing antioxidant?

    Radia Slimani

    you can also use the cytochrome C reduction assay for superoxide anion measurement

  • Frederick E Domann added an answer:
    Could NADPH dependent Nox activity be more sensitively detected in an EcSOD deficient background?

    Certain membrane associated Nox family enzymes (1,2 & 4) make superoxide, but extracellular superoxide dismutase (EcSOD, Sod3) removes superoxide. Therefore, tissue specific EcSOD deficient cells or organisms mice could be used to detect Nox derived superoxide with greater sensitivity.

    Frederick E Domann

    Thanks to all that responded so far !! The comments about INTRAcellular SODs are very important; thanks Rajesh and Just for that insight. And thanks Just and Igor for the suggestion of the chemiluminescence assay. However, as to the specificity of the chemiluminescence method for detecting membrane associated NADPH dependent Nox activity, that is problematic to me and many others and its use for this purpose has been recently challenged:

  • Martyna Elas added an answer:
    Has anyone experienced increased Dihydroethidium (DHE) but decreased mitoSOX red when treated with ROS-inducing compound?

    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.

    Martyna Elas

    Dear Han Shen,

    it may also depend on what type of drug you are using - where exactly the ROS are generated. If in the cytosol, or even outside cells, then the results you are getting is quite plausible. If you are expecting an increased mitochondrial respiration, then you should be getting an increase in Mitosox red fluorescence. 


  • Norbert Bittner added an answer:
    Can an increased level of hydrogen peroxide reverse the reaction of superoxide dismutase in an assay ?

    I'm having the problem that in my SOD assay (NBT assay, Pyrogallol assay) the sample reaction is showing a faster O2- production than the blank of the SOD standards. Means that the samples are way darker in color after 5min of reaction than the SOD blank. I'm working with Sephadex desalted pine crude extracts.

    Norbert Bittner

    So far none of the assays worked unfortunately... But thanks for your hints

  • Sourav Laha added an answer:
    I want to calculate the torsion angle between a superoxide and peroxide anion in GSAS. Anyone knows how it can be calculated in GSAS?

    I have ~200 tof neutron diffraction dataset (T-dep.). I have analysed the datasets by sequential refinements using GSAS. However, I could not figure out how i can calculate the torsion angle sequentially between the two vectors; superoxide and peroxide anions. I used Materials Studio which provides a tool to measure the torsion angle between these two vectors but i have to get an estimated error which is not provided in materials studio.

    Sourav Laha

    If you are using EXPGUI of GSAS pacakge,

    You can go to Results to geometry

    A question will appear 'Print input atoms? <Y/N>'  you can choose an option

    Next question will be 'Enter geometry option <some options>' you can type '?' to get the details of the options or directly type 'a' for computing dihedral angle and follow the steps.

    If you are using GSAS, there should be something similar but I am not familiar with it.

    Hope it will help you


    With regards

  • P. Pardha-Saradhi added an answer:
    What is the mechanism of superoxide anion free radical scavenging assay?

    what is the mechanism of superoxide anion free radical scavenging assay of plant extract?

    P. Pardha-Saradhi

    Protocols that are widely being used (that we also use) involve (i) generation of superoxide anion radicals (either by exposing riboflavin to light in presence of electron donors, or throught PMS-NADH system or  xanthine-xanthine oxidase system); and (ii) reaction of superoxide anion radicals with certaing molecules/biomolecules to yield a product that can be determined spectrophotometrically (production of nitrite in first case, formation of formazan in the second case and reduction of cytochrome c in third case); Higher the concentration of superoxide anion radicals more would the generation of nitrite, formazon or reduction od cytochrome C. Therefore, in presence of superoxide desmutase that uses superoxide anion radicals as substrate, one would expect decrease in the formation of nitrite, formazan or reduction of cytochrome C. Accordingly, higher the SOD activity lesser would be the formation of nitrite, formazan or reduction of cytochrome C.

  • Samuel Chuah added an answer:
    Can all members of Reactive Oxygen species induce breakage in Graphene Oxide sheets? (GQD production)

    Photo- Fenton reaction mediated fabrication of Graphene Quatum Dots is well recorded. Keeping in view of the oxidative action of the hydroxyl ions hence produced, I am curious about the ability of other reactive species (like that of superoxide ion, singlet oxygen) in breaking the GO sheets.


    Samuel Chuah

    From my limited knowledge on oxidation of GO, the attachment of oxygen functional groups to the basal plane of GO (epoxide, carboxyl, hydroxyl and carbonyl) does break the grapheme sheets

  • Nai-Kei Wong added an answer:
    Does anyone know the rate constant for the generation of superoxide by NOX enzymes please and if so the reference?

    Kinetics of redox enzymes

    Nai-Kei Wong

    Thank you for sharing. It's an interesting question with few references, in part because NOX is a complex requiring multiple subunits to function. The in vitro reconstitution work was an admirable effort. 

  • Micheal S Ward added an answer:
    Normalisation of OXPHOS by CS (ie complex respiration rates) is standard protocol. So should OXPHOS by-products (ie SO) also be treated as such?

    I am a diabetic complications researcher and I'm carrying out a number of measurements on freshly isolated mitochondria, such as respiration by Seahorse technology. I normalise this data by citrate synthase activity as I realise the enzyme is rate limiting in the TCA cycle for citrate production. Therefore I have been attempting to reconcile if normalisation of by-products of OXPHOS are also needed in the likes of superoxide, hydrogen peroxide which I have measured by MitoSOX and Amplex red assays respectively. I am also carrying out MnSOD activity assays for identification of possible changes in dissipating enzyme activities.

    Micheal S Ward

    Hi Alexander,

    Thank you for the response, I have downloaded your excerpt and will read through it as I have some of your work on other mitochondrial topics. I find your pieces of work to be quite frank and offering a facts based opinion that differs with deemed 'conventional' methods being highly published and fascinating which allows for my greater critical analysis.

    Thank you.

  • Emilia Wojtera added an answer:
    Does anybody know another form of generate superoxide radicals for superoxide dismutase determination diferent of xanthine and xanthine oxidase?

    High I need proof another form of generate superoxide radicals for superoxide dismutase determination with the reduction of citocrome C, that be different of radicals generate by xanthine/xanthine oxidase.


    Emilia Wojtera

    You can use hypoxanthine or xanthine, try with diazoxide, ET-1, AngII... 

  • Alexander V Panov added an answer:
    Does ambient proton concentration (pH) alter the behavior of ROS/RNS?

    Let me preface this by saying I am not a biochemist.   My logic may be very incorrect, but I'm looking for some guidance from someone who has a much stronger grasp on this field.    Or if anyone can point me toward an appropriate reference.   It's not an easy topic to google search; too many unrelated results.

    My question is more of a curiosity.  I am wondering whether the behavior/concentration ROS like super-oxide and others (the more reactive species) would interact with the ambient proton concentration in a cellular compartment.  To put this question another way, I'm wondering if decreasing the pH would tend to "quench"/reduce the generation of highly reactive and potentially damaging species.  

    Isn't this essentially what antioxidant  compounds do?   They function as proton donors?   I suspect there is a deeper level of information related to the energetic exchange that I'm not aware of.     

    To also ask the reverse question:  Would the biological generation of ROS/RNS (say, via mitochondrial dysfunction) alter the availability of protons/influence the local pH? 

    Alexander V Panov

    I would like to add another 5 cents to the current discussion.
    When discussing pH, we should remember several important issues: 1) pH is in most bulk environments, we usually consider cytosol and mitochondrial matrix, is highly buffered. In the matrix pH may remain constant even though the ΔΨ may drop significantly; 2) the mitochondrial matrix is a hard gel, therefore, if there are changes in pH they occur locally in a narrow space either near an enzyme, which produces or consumes a proton, or near the membrane; 3) The metabolic events, including production of superoxide radicals or their removal, which are coupled to other metabolic events, occur in the multienzyme complexes and proceed usually irreversibly.
    One of the most important species of ROS is the hydroperoxyl radical, also known as the perhydroxyl radical, which is the protonated form of superoxide with the chemical formula HO2. Hydroperoxyl is formed through the transfer of a proton to an oxygen atom. The concentration of O2 in the lipid phase of the membrane is 4-5 times higher than in the extra-membrane environment, and the superoxide anion is rapidly extruded from the membranes. The superoxide anion, O2•−, and the hydroperoxyl radical are in equilibrium in aqueous solution: O2•− + H2O is in equilibrium with HO2 + OH−. The protonation/deprotonation equilibrium exhibits a pKa of 4.88; consequently, about 0.3% of any superoxide present in the cytosol of a typical cell is in the protonated form. Although HO2 is also less hydrophobic and thus excluded from the lipid phase of membranes, nevertheless, it is more soluble in lipids than superoxide anion. I think that major formation of HO2 occurs not in the cytosol, but in the water-containing space near the superoxide-producing center inside the membrane. Unlike O2−, which predominantly acts as a reductant, HO2 can act as an oxidant in a number of biologically important reactions, such as the abstraction of hydrogen atoms from tocopherol and polyunsaturated fatty acids in the lipid bilayer. As such, it may be an important initiator of lipid peroxidation. The concentration of HO2 in the membrane may depend on local pH. I think that changes of pH in the bulk environments (cytosol or incubation medium) have little influence on reactions, which occur locally in the membranes and the membrane’s micro-environment near the enzyme’s center where superoxide is formed.

  • David Tapley added an answer:
    What is the best activity assay for Catalase & SOD activity in Cnidarian?

    I need a kit that is sensitive and not time consuming. Thank you.

    David Tapley

    I used several methods on cnidarian tissues in the past -- I ended up settling on the nitro blue tetrazolium method for SOD (Spitz and Oberly) and the modified (as in the Sigma catalog) method of Beers and Sizer for CAT. Message me with your email and I can probably dig up my old protocols and send them to you. 

  • Daniel Medeiros Lima added an answer:
    What is the best method for quantify ROS in heart sections?

    What of method is more specific to measure ROS in cardiac sections of rats? DHE is not very specific, however I see many publications using the method.

    Thank you for your help.

    Daniel Medeiros Lima

    Thanks Adam and Karel, their information were of great help.

  • Ryan D. Taylor added an answer:
    Any suggestions on BMPO Spin Trap Hydroxyl/Superoxide Adduct formation rates?

    I am looking for information on the formation rates of two radical adducts BMPO/OH and BMPO/OOH. There are many studies on the formation of these radical adducts using xanthine oxidase or Fenton reactons but there does not appear to be anything on competitive reaction rates (cases where the OH radical and OO- radical are competitively reacting with BMPO). 

    Any insight would be greatly appreciated. 

    Ryan D. Taylor

    Thank you for that great information Deborah! I really appreciate the insight.

  • Tarana Arman added an answer:
    What is the minimum amount of DHE (Dihydroethidium) that could be used to detect the superoxide ions in saccharomyces cerevisiae?

    I have induced a chromium stressed condition for the yeast culture and want to assess the ROS increase in them. However, I am unable to decide upon the upper and lower limits for the amount of DHE to be used.

    Tarana Arman

    Thanks Philippe, your answer was indeed helpful.

  • Mustafa Yavuz Köker added an answer:
    Does CD40 expression on B cells require reactive oxygen species?

    CD40 expression on B cell has superoxide dependent pathway? So since CGD patients lacks superoxide production do they express CD40 on B cells?

    Mustafa Yavuz Köker

    NADPH oxidase activity order Neutrophil>monocytes>eosinophil>dendiritic cell>B cell,

    For CD40, I think yes. But it is very complex, need pathway analysis. CD40 deficiency is another immunodeficiency. NADPH oxidase modulate it, but does not dependent. I think.

  • Karel Chalupsky added an answer:
    Does anyone know of a protocol for qualitative assessment of the presence of superoxide dismutase in bacteria?

    I would like to screen my bacterial isolates for their ability to detoxify superoxide through superoxide dismutase. I would like to know of any rapid qualitative test to assess if my isolates have this enzyme. Most of the searches I have initially done involve spectophotometric analysis.

    Karel Chalupsky


    25 µM cytochrome in PBS was incubated for 5min at 37 C with addition of KO2. The absorbance was read spectrophotometrically at 550 nm against distilled water blank. Reduction of cytochrome c in the presence of SOD (100U/ml) was subtracted from the values without SOD. The absorbance differences between comparable wells with or without SOD were converted to equivalent O2•− release by using the molecular extinction coefficient for cytochrome c of 21 mM−1 cm−1

    Superoxide dismutase
    30000Units (sigma # S-8160)
    Stock 30000U/ml (30U/ul) then freeze aliquots of 50ul
    30 U/ul so 50ul =1500U then add 250 ul water ->1500 U /300 ul = 5 U/ul
    dilute above 1 : 4 with PBS so have 1 U/ul -> then 60ul = 60 U

    Add 60ul of SOD to a well of 6-well plate containing the 480 ul PBS (sample)
    the total volume will be 600ul so the conc. of SOD is 100 U/ml

    Cytochrome c partially acetylated (Sigma # C-4186)
    Cytochrome c from hourse heart ( # C-2506)
    6.19 mg cytochrome / 2 ml = 3.1mg / ml = 250uM
    Solution is only good for a few hours!
    Light and oxygen sensitive keep solution in the dark and covered!

    Add 60 ul of cytochrome c to sample solution – final volume 600ul (final c of cytochrome c is 25uM)

    Read absorbance at 540, 540 and 560nm

    To calculate the cytochrome c absorbance use the pathlenght corrected values only:
    Take average of the readings at 540nm and 560 nm (540nm + 560nm)/2
    From the readings at 550nm subtract this average 550nm –[( 540nm+560nm)/2]
    This subtracts the baseline from absorbance 550nm peak for reduced cytochrome c

    Potassium superoxide solution (3mg in 1ml) in absolute DMSO was used as superoxide donor. Due to low solubility and possibly water content in DMSO, the real concentration of superoxide anion was detected by cytochrome c reduction and calculated using molecular extinction coefficient for cytochrome c of 21 mM−1 cm−1 .
    USE xanthin oxidase system as superoxide generation system. It is more reliable and physiologic.

  • 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

    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

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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