For all scientists involved in oxidative stress research.

• Sri Trisha

  Will cell lysate prepared by RIPA buffer be ok for GSH, SOD assays?

  I would like to check GSH, SOD activity from cell lysates. Can I use cell lysates stored at -20C prepared by RIPA buffer for western analysis?

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  • 
    Md Wasim Khan replied

    Sure you can use that. What is the concentration of your cell lysates? Newez RIPA doesn't hinder any antioxidant enzymes mediated catalysis. Which method are you using to determine GSH and SOD?

    3 days ago
• Sri Trisha

  Do flavonoids have ROS scavenging activity?

  We have partially purified a herbal extract (which contains a mixture of flavonoids) in which we found anticancer properties. Further, we checked intracellular ROS generation doses dependently. IWe have partially purified a herbal extract (which contains a mixture of flavonoids) in which we found anticancer properties. Further, we checked intracellular ROS generation doses dependently. I doubt that flavonoids are generally ROS scavengers, but in our case they are producing intracellular ROS in cancer cells. Could any one explain this to me?

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    Sri Trisha replied

    Thanks Seyed for proper explanation. even i found articles regarding this.some are here. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 18, pp. 13468–13476, May 4, 2007. THE JOURNAL OFThanks Seyed for proper explanation. even i found articles regarding this.some are here. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 18, pp. 13468–13476, May 4, 2007. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 36, Issue of September 9, pp. 31498–31507, 2005 . Toxicology in Vitro 26 (2012) 16–23. from this i came to know Antioxidants can be ROS producers in turn induce apoptosis in cancer cells.

    5 days ago
• Łukasz Ołdakowski

  How to get 10mM of 2,4-dinitrophenylohydrazine?

  I have to prepare 10mM of DNPH. The commercialy avilable chemicals are for example here http://www.tci-uk.co.uk/catalog/D0845.html and they usally contain about 30% of water (it is explosive whenI have to prepare 10mM of DNPH. The commercialy avilable chemicals are for example here http://www.tci-uk.co.uk/catalog/D0845.html and they usally contain about 30% of water (it is explosive when dry) How much of powder do I have to add to 2M HCl to get 10mM DNPH? Do you know good manufacturer of DNPH. I want to measure protein damage in mammalian tissues. I will be grateful for help.

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    Łukasz Ołdakowski replied

    Thanks for information

    10 days ago
• Natalia Sh

  Oxidative stress analysis

  Can isolated red blood cells be used for oxidative stress analysis following incubation with an oxidation compound? (in-vitro oxidative stress analysis in red blood cells)

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  • 
    Leader Nice replied

    Thanks for all your comments. thank so much for all.

    11 days ago
• Sri Trisha

  Should H2DCF-DA be applied pre- or post- drug treatment for the best results?

  I am trying to determine ROS production by using the dye H2DCF-DA fluorochrome with suspension cells. I tried treating the cells with the drug before wahing and adding 20uM H2DCFDA in PBS. I thenI am trying to determine ROS production by using the dye H2DCF-DA fluorochrome with suspension cells. I tried treating the cells with the drug before wahing and adding 20uM H2DCFDA in PBS. I then washed once more and re-suspended in PBS. FACS results show the control with a greater peak shift than the h202 50uM positive control. In another case I just added H202 in the end and there was a good shift in this case without a wash step. Could anybody help me with this protocol?

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  • 
    David Kessel replied

    We have never had a problem with phenol red. It can be omitted since the only purpose is to show that the pH of the medium is OK (not alkaline which means not enough carbon dioxide and not acid whichWe have never had a problem with phenol red. It can be omitted since the only purpose is to show that the pH of the medium is OK (not alkaline which means not enough carbon dioxide and not acid which means too many cells. It is improtant to add the DCFDA FIRST or you will miss all of the transient ROS. Finally, DCFDA is not the best fluorescent assay since it can be auto-oxidized. We prefer DHR (dihydrorhodamine 123) which is more specific for hydrogen peroxide and OH radials.

    13 days ago
• Carlo Cervellati

  F2-isoprostanes are widely considered as the most reliable marker of oxidative stress, at least when GS-mass spectrometry is used to detect them. Can it be considered a biomaker of oxidative stressF2-isoprostanes are widely considered as the most reliable marker of oxidative stress, at least when GS-mass spectrometry is used to detect them. Can it be considered a biomaker of oxidative stress or only of lipid peroxiation? The fact that it derives only by peroxidation of arachidonic acid is an intrinisc limitation, is not it? Most probably we can say that F2iso are specific markers for lipid peroxidation, but what about the sensitivity in the detection of overall oxidative damage?

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• Lakshmy Ramachandran

  Is there any relevance in checking the reduced glutathione levels in an in vitro setup to measure the oxidative stress status?

  Is there any relevance in checking the reduced glutathione levels in an in vitro setup to measure the oxidative stress status? For example- I work on radiation induced oxidative stress (in vitro/ inIs there any relevance in checking the reduced glutathione levels in an in vitro setup to measure the oxidative stress status? For example- I work on radiation induced oxidative stress (in vitro/ in vivo). Under in vitro conditions, one of the assays that can be relied on for measuring the extent of oxidative stress is determining the lipid peroxidation level. Likewise, is it appropriate if I assay for the reduced glutathione levels or any other endogenus antioxidant enzyme levels such as Glutathione peroxidase, Catalase etc to determine the oxidative stress status??

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  • 
    Harald Schmidt replied

    Hmm, instead of dialogs or conversations, there seem to be strings of monologs going on here.

    22 days ago
• Petr Boucek

  Use of urine 8-isoprostanes as marker of oxidative stress increase during an acute kidney clinical event?

  Does anybody have an experience with a significant change in spot urine 8-isoprostanes (iPF2α- VI EIA; pg/ mg creatinine) following an acute clinical event concerning the kidneys (e.g. acute renalDoes anybody have an experience with a significant change in spot urine 8-isoprostanes (iPF2α- VI EIA; pg/ mg creatinine) following an acute clinical event concerning the kidneys (e.g. acute renal failure of various origin) as proof of increased oxidative stress?

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  • 
    Petr Boucek replied

    Thanks a lot, best regards

    25 days ago
• Ram Kumar

  What are the steps involved in estimating brain Arsenic levels?

  Quantification of arsenic levels in the brain.

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  • 
    Rajesh Yadav replied

    Arsenic levels in individual brain regions exposed to arsenic can be estimated by atomic absorption spectrophotometer (HSAAS, ZEEnit 700) following the method of Ballentine and Burford, (1957).Arsenic levels in individual brain regions exposed to arsenic can be estimated by atomic absorption spectrophotometer (HSAAS, ZEEnit 700) following the method of Ballentine and Burford, (1957). Briefly, 2ml of concentrated nitric acid followed by 0.5 ml of perchloric acid was added to 100 mg of tissues. The sample was then digested over a sand bath until the solution becomes yellow in colour. If the colour of the digest was brown, more nitric acid and perchloric acid were added and the oxidation was repeated. The digest was made up to known volume with deionized water. Aliquots of this were used to estimate arsenic using atomic absorption spectrophotometer. A calibration curve was constructed by adding known amounts of arsenic standard to calculate arsenic levels in the brain regions.

    25 days ago
• Vishwa Bandhu Chaturvedi

  What will be impact on oxidative stress by increasing air uptake by deep breathing?

  Oxidative stress

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  • 
    Vishwa Bandhu Chaturvedi replied

    What are the possible effects of lower carbon dioxide and higher oxygen levels of lungs and blood.what will be the possible biochemical changes at various target points especially at mitochondrialWhat are the possible effects of lower carbon dioxide and higher oxygen levels of lungs and blood.what will be the possible biochemical changes at various target points especially at mitochondrial levels( normal body cells and brain and nervous cells) and other target points .

    26 days ago
• Rachel Ching

  SOD and catalase measurement in rat's brain

  Hello, I am Rachel, a PhD student in HKU. I would like to measure SOD and catalase activites in rat's brain. I have bought commerical kits from Cayman chemical company (96-well plates, item # :Hello, I am Rachel, a PhD student in HKU. I would like to measure SOD and catalase activites in rat's brain. I have bought commerical kits from Cayman chemical company (96-well plates, item # : 707002 and 706002). However, I am not sure how many grams of tissue and how much homegenized buffer shoud I use for tissue homogenization for each assay. Also should the sample be diluted before doing the assays? Since the materials are limited, it is not possible for me to assay the sample at several dilutions before doing the large-scale measurement... Thank you so much!!!

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  • 
    Paul Ernsberger replied

    The Cayman kits are easy to use. Probably you will have to use a trial and error method to get the right concentration of protein, because enzyme levels vary considerably in tissues.

    26 days ago
• Cherif Abdenour

  metal detoxification

  I started thinking about how to find natural plants for metal detoxification

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  • 
    William Jackson replied

    Coriandrum sativum - fresh aerial parts of coriander - (chinese parsley/cilantro/danye.) http://duckduckgo.com/?q=cilantro+chelation&t=lm

    27 days ago
• Diaa Ragab

  What are the steps for the assay of MDA in tissue by TBARS-based method in detail?

  I also wish to know the concentrations & methods of preparation of the solutions used in the assay.

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  • 
    hossam mohamed Omar replied

    LPO products as TBARS were determined according to the method of Ohkawa et al., (1979) as follow: Procedures 1- Large glass tubes were labeled for each sample and standard (TMP). 2- To each tubeLPO products as TBARS were determined according to the method of Ohkawa et al., (1979) as follow: Procedures 1- Large glass tubes were labeled for each sample and standard (TMP). 2- To each tube the following: 0.1 ml sample, 0.1 ml sodium dodecyl sulfate (SDS), 0.75 ml acetic acid, 0.75 ml thiobarbituric acid (TBA), 0.3 ml distilled water were added and mix. 3- Tubes were heated for 60 min in a boiling water bath (95°C), after cooling in tap water, 0.5 ml distilled water and 0.25 ml n-butanol/ pyridine were added and vortex. 4- All tubes centrifuged at 3000 rpm for 10 min then 2 ml of the organic (top) layer were removed and placed in 1.5 ml in quartz cuvette and measured spectrophotometrically at 532 nm against n-butanol/ pyridine as a blank. 5- The level of lipid peroxidation was expressed as nmol of malondialdehyde (TMP)/mg protein

    27 days ago
• Heidrun Karlic

  Oxidative stress is an important topic

  Regarding current research in cell biology of leukemias from the elderly, I am highly interested in ROS...

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  • 
    Nancy Hart replied

    Thank you for the references. Very interesting to consider in light of oxidative stress and proposed mechanisms.

    27 days ago