Easier than DAB staining (http://www.bio-protocol.org/wenzhang.aspx?id=263) or do you mean in a more quantitative way? Please give some details.

It is possible to measure carotinoids with resonance Raman

spectroscopy (Jürgen Lademann et al.) optically from the

human skin. The device is being used as stress sensor.

Regards,

Joachim

Yes. Try to use MTT coloration. Originally they use it for measuring cell vitality. But I tried it with overall free radicals accumulation and it works. It is a spectroscopic measurement, very simple.

Simply you can use Dichorofluorescein dye to measure ROS generation and quantification.

Add this dye to the sample to be checked for ROS generation. This dye will be converted to non-fluorescence to fluorescent compound in presence of ROS. The fluorescence measurement at 524nm will quantify the presence of ROS in sample.

Good Luck..!!

A simple method is fluorometric determination using DCFH-DA. There are some important considerations like assay (incubation) has to be done in dark.

AM not sure about MTT, primarily for two reasons.

1. Its non-specific when it comes to what it receives electrons from-mitochondrial dehydrogenases, dt-diaphorase etc can transfer e- to MTT.

2. The formazan is insoluble, hence it needs to be dissolved or at least stabilized using gelatin.

The 2'-7'dichlorfluorescein diacetate (H2-DCFDA) method of measuring ROS generation is very simple, and being fluorometric it offers very good sensitivity. If you have a fluorometer capable of kinetic measurements you can also do a time-dependent assay quite nicely.

The method referenced here is quite simple: Ferrer E, Juan-Garcia A, Font G, Ruiz MJ. Reactive oxygen species induced by beauvericin, patulin and zearalenone in CHO-K1 cells. Toxicology In Vitro 2009;23:1504-1509

I hope that helps.

Just a side note. Tobias Dick from German Cancer Research Center

et al. discovered that oxidized glutathione is enclosed in vacuoles,

and that may lead (or have led) to wrong measurements

in studies.

http://www.dkfz.de/en/presse/pressemitteilungen/2012/dkfz-pm-12-66-Preventive-Detention-for-Oxidizing-Agents.php

http://www.dkfz.de/de/presse/pressemitteilungen/2012/dkfz-pm-12-66-Sicherheitsverwahrung-fuer-Oxidantien.php

Regards,

Joachim

Dear All,

Thank you very much for your timely and valuable suggestions, i am looking forward to your suggestions.Somebody suggesting to use histidine. tryptophan or ADPA to measure ROS, but i am not sure about it? do you have any idea about it?

MTT is exactly for determining the overall free radicals accumulation; for the specific radicals there are other markers. The determination may be done in a way that permits to measure the formazan formed. If the free-radicals formation is too intensive, DMS might be used as a solvent. The non-specificity of MTT is an advantage if the total free-radicals accumulation is evaluated:)

Mtt for ros? Would love to try. Pls share reference for protocol.

In this case, two methods can be employed.

Direct method is to use MTT reduction assay.

Indirect method would be LDH release assay.

by co-relating the results obtained by these assays you can confidently defend your results.

Medicinal Chemistry, vol.4(4), 371-378, (2008) - for assassmento of the effect of a compound on the oxidative status of blood plasma - ex vivo

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Trakia Journal of Science, vol.10(1), 331-335, (2012)- for assessment of oxidative status of tissues after in vivo experiment.

I apologize for the very poor quality of the latter, but the book was very thick. I am going to skan better copies and I will supply them in my profile.

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If i have understood the question correctly, a method for measurement of reactive oxygen species is being sought. Its a very specific measurement and hence a specific question.

Ldh leakage is an assay often used as surrogate marker in cytotoxicity assessment. Not sure how ldh can shed on levels of ros.

It will be interesting to see if mtt can be reduced in the presence of only h202 and Fe2+.

Hi, Kalpana, do you want to measure intracellular or extracellular ROS? Which kind of ROS: superoxide or hydrogen peroxide? The method you have to used largely dependent from the questions you ask. For the intracellular ROS accumulations you can use a couple of fluoresencet dyes like DHE (superoxide), H2DCFDA for hydrogen peroxide), in some case NBT for superioxide is also good, but with many precautions. For the extracellular ROS you can use DHBS/AAP method which give you quantitative results, or Oxy-Burst, which give you only localization.

Overall, one can not ,easure ROS generation, but ROS accumulations, ea. differences between production and scavenging.

Good luck!

I am using Cell Rox (Invitrogen) with great results. You can easily adpat this method for flow cytometry, plate reader or fluorescence microscopy. Also, you can choose the dye (red, green, orange) which is useful if you are using GFP+ cells, for example.

Good luck.

For whole cell ROS you may use DHE, for mitochondria use Mito-SOX, under fluorescent microscope you may quantify superoxide formation. Similarly use DCF for H2O2 but ONOO- can also react with DCF. Or, you can use Amplex-Red and plate reader for H2O2 formation. Lucigenin 5 mcM for superoxide or luminol for H2O2 can be used for relative real time measurement under luminometer. These are the few methods I have used and hope they can help you.

Simple methods means you have to use biochemical assays. Apart from MTT, fluorimetrically ROS can be measured using scopoletin method or using HRP mediated homovallinic acid (HVA) oxidation method. Both the above are opposite in their principle of measurement. You should get lower fluorescence value with scopoletin and higher value with HVA at higher ROS leevel. More specifically, both the above are specific for H2O2 estimation. For supper oxide level, you can go with NBT method. Probably no specific method exists for OH estimation. Phenol oxidase method is also another spectrophotometric method to measure H2O2. Spin trapping method is the most reliable and advanced one to measure ROS.

Be aware the method you plan to use when submit for publication. Recent trend is to use either HPLC or EPR. Other method can easily be criticized by reviewers in high IF journals.

I would say EPR spectroscopy with DMPO or BMPO. You can easily know what ROS has been produced, even on cells. See this publication : http://www.pnas.org/content/81/23/7269.full.pdf

ROS production can be easily assessed by using H2DCFDA and analysing samples using flow cytometry.