Questions related to Lipid Peroxidation
I would like to test for lipid peroxidation (ferroptosis-induced) in leukemic cells using flow-cytometry analyser BD FACSCanto. Now on the analyser, which parameters do I use for analysis of lipid peroxidation?
I want to include AA/Vit. C as a positive control for my antioxidant study using Vero cells to compare with my drug compounds. I will measure MDA (lipid peroxidation marker), SOD, and Catalase activity.
- How do I prepare the stock solution of AA and what are the proper storage conditions?
- What is the usual concentration of AA used for in vitro study?
- I read AA is light sensitive and unstable in an aqueous solution, so if I add AA in culture media as treatment and incubate 12 to 24 hours, will it work? How to solve this problem?
I used the brain, plasma and serum sample of rats. Preparation of the samples were done on ice bath and centrifugation done at 4 C, then the supernatants were kept in -20 C. the test was done with in 7 days. The brain samples gave satisfactory results on lipid peroxidation test (using 20% TCA and 0.67 TBA). However, plasma and serum samples did not produce any color at all, what cause the absence of colour in these samples?
ps: I also have tried different preparation for the plasma, using citrate and EDTA.
I have unexpected results in my project where the positive control group had decreased lipid peroxidation levels in comparison to the negative control group. I was thinking that a way to explain was considering that my study was in adult mice and the endogenous antioxidant enzymes are strong enough to hold agains the ROS, also the administration was subcutaneous.
I'm considering which assay to use for measuring lipid peroxidation in the neuroblastoma cell line. I came across these 3 options: TBARS, MDA assay, and ELISA. TBARS seems less specific compared to MDA assay and ELISA, because TBARS detects anything that reacts with thiobarbituric acid, whereas MDA and ELISA reacts only to Malondialdehyde.
However, I found it strange that from the papers I read, it seems that TBARS is commonest, followed by MDA assay, then lastly ELISA? I was wondering if this observation that TBARS is commonest is true, and if so what's the reason behind? Is it just the price? But I don't find TBARS kit being a lot cheaper than MDA assay and ELISA... Is it then the easy of operation, and the consistency of results?
I would also be grateful for any tips while working with lipid peroxidation / oxidative stress assay, assay recommendations and the like.
Thanks in advance for your suggestions.
Lipids are susceptible to oxidation when exposed to free radicals. Polyunsaturated fatty acids are particularly vulnerable to lipid peroxidation resulting in cell damage. What are the harmful effects of lipid peroxidation on health?
This dye itself has color of PE-Cy5 and after interaction with the lipid ROS, it emits green fluorescence. I have also found that this dye also interferes with the PE, PE-Cy7 channels somewhat. So I was wondering if there is an alternative to this dye? (Also, why is this the standard dye for ferroptosis related lipid peroxidation?)
I have searched some research articles related to Ferroptosis. Many kinds of experiments have been done. like, staining, GSH assay, lipid peroxidation assay, etc.
1. What methods (both basic and confirmatory) should be followed?
2. Most of the researchers have been used in the kit. Any method is available other than using the kit?
i did this protocol to measure the anti-lipid peroxidation activity of plant extracts.
The lipid peroxidation assay was carried out using the modified method of Ohkawa et al. Briefly 100 μL of bovine brain extract sigma (B1502) was mixed with a reaction mixture containing 30 μL of buffer, (100 μL) of (1- 1000 ug/ml) extract or positive control vitamin C , and 30 μL 5 mM sodium nitroprusside (SNP) as the prooxidant. The volume was made up to 300 μL by water before incubation at 37°C for 2 hrs. The colour reaction was developed by adding 300 μL 8.1% SDS (sodium dodecyl sulphate) to the reaction mixture, this was subsequently followed by the addition of 500 μL of acetic acid (pH 3.4) mixture and 500 μL of 0.8% thiobarbituric acid (TBA). This mixture was incubated at 100°C for 1 hr. Thiobarbituric acid reactive species (TBARS) produced were measured at 532 nm.
The problem is that the generated pink colour of chromogen increases with increase the positive control concentrations (vitamin C). where the control the brain extract + sodium nitroprusside (SNP) as the prooxidant gives the lightest pink colour.
Thanks in advance...
We stain our leaf samples of control and stressed with Schiff reagent. However, I did not get good results.
Please suggest other staining methods for lipid peroxidation.
Hi, I am working on HK-2 cells and want to check for MDA accumulation after treatment with our compounds of interest. I am using MDA assay kit from Sigma. However, the kit does not mention about whether the lysate can stored or not. I wanted to know if we can store the cell lystae in MDA lysis buffer of the kit. If yes, at what temperature and for how long? Also, can we use this lysate to measure protein for normalization across samples?
I am analyzing clam tissues for lipid peroxidation using the TBARS protocol. To extract lipids for MDA analysis, is it necessary to use PMSF when I am not interested in protein analysis?
I'm looking for adaptations of Hodges et al., 1999 essay for MDA quantification to microplate, has anyone aware of this? The initial paper is naturally in cuvettes, and most papers don't go into much detail about their methods, becoming dificult to know if this is being done or I'll just have to try it for myself.
Cheers, Luís Pereira
PS: My target are brown seaweed, in case anyone has specific opinion about this.
Quantifying the level of Malondialdehyde (MDA) is a well established method for detecting lipid peroxidation. Sometimes data is expressed as MDA produced per mg or gram protein. Since we are measuring the level of an aldehyde (MDA), then what is the reason to express the date in per mg or gram protein.
I am looking for a skilled personnel in Kuwait University that is familiar with antioxidant studies using ELISA kit or other techniques on rat's brain homogenate, particularly the hippocampal and anterior cortex regions.
I followed the specification of Lipid peroxidation (MDA) assay kit from Sigma, and performed MDA assay. But I did not get any pink-color, I got some white sediment instead. Do any of you have the same problem or do you have any idea about that? The MDA standard worked and I can see pink-color, so the procedure of my protocol should be fine.
100% followed the protocol from Sigma. I harvested cells in 10cm dishes.
Hello everyone! I want to test if my synthesized compound inhibit reaction of lipid peroxidation. I have searched for protocols in published literature but aren't clear enough since specific time was not mentioned. I found methods where sodium linoleate and AAPH were used. Is that suitable method or can I determine inhibition by TBARS method? Thank you in advance
I would like to measure MDA levels on keratinocytes using a lipid peroxidation assay, I have searched for protocols in published literature but aren't clear enough since I have never performed this assay. Also, if anyone has performed this assay, can you provide me with the catalog number of the kit you have used?
Thanks in advance,
Synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), have been utilized to reduce lipid peroxidation in food products. However, I couldn't find a report on these chemicals as active compounds to reduce oxidative damages in human body when the living organisms unable to develop their own antioxidant defense mechanis.
I am conduction some bio-assays on the digestive tracts of bivalves exposed to a series of concentration of the same chemical, along with keeping control samples (cannot mention chemical or detailed results due to data concerns). In short, after testing and statistical validation was completed we have an odd result:
Glutathione S-transferase (GST) assays show samples had no statistical difference in activity between control samples and any samples exposed to the chemical, and only a mild rise in average activity that can't be thoroughly distinguished between control and chemical exposed samples. Meanwhile other tests (Lipid peroxidation, DNA damage, FRAP, etc) all show a clear difference in response, with the chemically exposed samples all showing rises in stress responses. This result seems quite odd, as GST is often the starting indicator of whether there is any chemically induced oxidative stress, and the next few assays often help to pin down where and why the stress is being caused. I know that this assumption (GST = all stresses) is not guaranteed but it is fairly common practice to use it as such.
My only real idea is that in general the mussels remained stressed even after 14 days of lab storage to purge at optimal temperatures, media cleaning and regular feeding. And as such the GST remained raised on all samples from general stress from their new environment, but then the other assays showed how the added chemical distinctly rose the stresses in more specific ways. Does anyone else know of other possibilities as to why GST would remain indistinct between control and chemical exposed samples, while other assays would show a clear rise in stresses in chemically exposed samples?
I tried to run a lipid peroxidation assay with DPPP , the plate in black with clear bottom. I read the fluorescence from top and since my samples are diluted in PBS I used put it in one well and use the read as blank. But everytime I read the plate I have different results. Is there any special consideration for running this type of essays in terms of wells localization? time?
I would like to analyze lipid peroxidation (TBARS COLORIMETRIC METHOD) in plasma samples of rats. But I don´t getting satisfactory results with the current technique that I possess. So, I'm looking for an alternative technique for the colorimetric spectrophotometric performance of this analysis in a 96-well microplate.
Hello, I am trying to induce oxidative damage in neurons (derived from IPSC) by using hydrogen peroxide.After treatment I want to see if there is any evidence of lipid per oxidation (by looking for malondialdedye) or if there is any evidence of 8oxoguanine accumulation. can anyone advised how long I should wait after treatment before assessing for these?
I am studying the mechanism of lipid peroxidation in tomato fruit. It would be appreciated if anybody help me sharing the detailed procedures of measuring MDA, hydrogen peroxide and superoxide content in fresh tomato tissue using spectrophotometer.
I want to do a fluorescence measurement (96 well) after a lipid peroxidation assay on Mia PaCa-2 cells using c11-BODIPY. Could anyone share a protocol with me.
Do I have to pretreat the cells with BOBIPY before my peroxide inducing agent or vice versa.
I am currently doing analysis of antioxidant enzyme activities in rat liver tissue. I would be glad to get current reference on antioxidant enzymes and lipid peroxidation assays
I am currently attempting to run a TBARS assay on algal samples under different stress conditions. I have been following the Hodges et. al, 1999 protocol from their paper, Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. I am using a spectrometer to measure the MDA equivalents but I am having difficulties getting the end quantitative values for all absorbance readings. When i perform a technical replicate on the same cuvette my absorbance values changes quite drastically. They usually increase but some do decrease. Is this reaction light sensitive? Am i detecting other compounds which increase in response to the spectrometer? I don't know how to get around this. Thanks in advance
We are trying to estimate lipid peroxidation in leaf tissues of the seagrass Phyllospadix torreyi, following the protocol from Hodges et al. 1999. But this protocol does not work (i.e. we do not obtain the characteristic pinkish-red chromagen). This is so weird, because we have used this protocol with other seagrass species and land plants, and it always worked in the past!. Furthermore, at the same time, we are using the same protocol and reactives for seaweeds in our lab, and we are obtaining fantastic results. Could anybody kindly provide me any explanation or could suggest some modification for this protocol? Thank you very much!
I want to determine MDA content in tomato leaves under salt stress conditions. For this purpose I have collected leaf samples after completion of different levels of stress treatments as well as foliar spray of amino acids. I preserved the leaf samples in 10% TCA and kept in freezer at -40 since 2 weeks. Now, when i started my experiment by following Health & Packer protocol, I am not getting any color at the end of reaction. All reaction mixture are transparent not showing any color (must be some shade of pink) when we put at water bath for 25 mints. Firstly I was thinking that may be some issue in samples than I got some fresh leaf samples and perform the same procedure but no result . All the chemicals used here are new so I am not getting the point? Could anyone please suggest me that what can be issue and guide me. Thanx
I am looking for a method to isolate and analyze reactive oxygen species (ROS)-damaged proteins. I know that there are kits to detect individual ROS such lipid peroxidation, protein carbonylation and others. But, I am searching for a method to combine all of these in one! Have you heard of such a method?
Any suggestion what I should change?
I'm trying of 2kV/cm ; 4 kV/cm; 10 kV/cm PEF treatment. What concentration of cells I should use?
During the analysis of lipid peroxidization (MDA assay) as a stress bio marker under abiotic condition in microbial cells by manual method -
1. is it necessary to carry out the Standard curve ?
2.If yes, MDA (commercially available) should be used as a standard or any other alternative standard is available?
3. Which solvent is to be used for dissolving TBA - a. TCA or b. NaOH ?
4. how to calculate (formula) the concentration of MDA in the sample after assay ?
I'm hoping to use the same (currently frozen brain) sample to measure GSH/GSSG/Total as well as MDA assay - what would be the best way to prep this sample?
I'm using the sigma kit for MDA and they suggest homogenizing in the lysis buffer containing BHT + Perchloric acid (150uL, 2N/10mg tissue).
The GSH/GSSG/Total kit suggests using 5uL of 6N perchloric acid/10mg tissue.
Could I potentially prep the sample as for MDA assay and use the same supernatant to measure glutathione too?
I have read that for lipid peroxidation we can use DPPP, alexa fluor 488 or Bodipy 581/591 C11, but I can't find specifically for Saccharomyces cerevisiae.. Can I use the same dyes for them? Maybe there are some articles about that?
hi, i need recent references for the estimation of Collagen which is similar to the protocol described by Woessner (1961), DNA similar protocol described by Burton, (1956), Lipid peroxidation similar to Buege and Aust (1978) and western blotting.
To perform Lipid peroxidation inhibition assays in egg homogenates, I have faced difficulties to make proper egg homogenate. It was difficult for me in pipetting the sample. Please somebody guide me the proper way to prepare 10% (v/v) egg homogenate.
I’m planning to investigate the toxicity of MDA for cell line. Has anyone know how I can synthesis Malondialdehyde & analysis the MDA standards by TBARS. Also, is there any book has related information's with MDA.
My reagent information is:
TMP: MW=164.20 g/mol, d= 0.997g/mol, 99%.
TBA: MW= 144.15 , 98%
whole antioxidants are required. However protocol which include the majority or main antioxidants are preferred.
In such diseased conditions I tried to examine the effect of plant extract and I would like to assess the changes in ACh concentration.
How can I prepare the standard curve of Malondialdehyde (MDA) especially with Tetraethoxypropane (TEP) for TBARS calculation in UV spectrophotometer. Can anyone suggest how should I prepare the standard solution of TEP and in what range ?
Now, i try to assess the MDA and antioxidant levels like SOD, gluthaione and catalse in human sperm. I used 0.01% Triton-x and centrifuge for 6000 g, then collected the supernantant to assess the MDA levels. But the MDA level it very low (less than 2 nmole). Has anyone recommend the best protocol to isolate supernatant in human sperm? Thank you for your kind suggestion.
I have synthesized magnetite nanoparticle and coated them with protein to render biocompatibility, I have already performed MTT assay and now planning to go ahead with ROS detection assay? How should I perform the assay, by detecting lipid peroxidation or is there any other source?
I have plant treated with different concentrations of copper. Interestingly, the callus exhibited increased levels of MDA, despite enhancement in their biomass. There was stimulation in some antioxidant enzymes such as SOD, POD and GST. How can you explain this? Have you faced this before?
Most paper that discuss heavy metal mediated-oxidative stress, particularly copper as a transition metal, relate the inhibition in growth or biomass to the increase in lipid peroxidation concomitant with inhibition or even stimulation in the antioxidant defense system, in contrast to my results.
I know that among the strategies the plant may employ is to repair the degraded membranes and refold the unfolded or misfolded proteins, but I am not sure if that is the situation for my samples. If it is, should it appear as decrease or increase in lipid peroxidation?
I did antioxidant analysis which was catalase. My samples were rats liver. How can i proceed to analyse the end results if the absorbance rate was more than 1.2 (as suggested in the kit manual)? The manual suggested to dilute the samples, however all the reagents were almost finished. Need advice on this. Many thanks for your help.
In paper or techniques manual, 7.8 value is taken for calculation of TBARS (mg malonaldehdyde/kg sample). But no one is mentioned how this 7.8 value is considered for calculation. Can anyone enlighten me it properly?
I have to measure the oxidative stress enzymes activities like SOD, Catalase, GSHpx and lipid peroxidation of workers having exposure to chromium from erythrocyte lysates, please recommend me some good protocols how to prepare human erythrocyte lysates?
We have measured the activities through enzymatic or non enzymatic assays of Super Oxide Dismutase (SOD), Catalase, Glutathione and lipid peroxidation from hemolysate, now if we need to calculate the same in serum, do we need to follow the same protocols or there are different protocols for serum. Please help to understand this.
I know that there exists a direct relationship between lipid peroxidation and SOD, Cat, GPX etc., But there is lot of ambiguity when it comes to working with a particular cell line. Please post your inputs here. Thank you.
I am developing a method to measure antimicrobial death caused by ROS in a bacterial culture.
In particular, I want to measure oxidative stress compared to my reference sample.
I thought of different fluorescent reagents, as would the H2DCF, HPF or Click-iT lipid peroxidation kit- imaging Alexa Fluor 488
I do TBARS assay by following the protocol of Ohkawa et al, 1979 (Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction). I need a clarification on the working protocol this method.
The following is the protocol as per original Ohkawa Paper, i.e.
1. 0.2ml of sample is added with 0.2ml of 8.1% SDS, 1.5ml 20% acetic acid at pH3.5, 1.5ml of 0.8% of TBA
2. This mixture is made up to 4ml and boiled for 1 hour at 95-degree celsius.
3. After cooling, 1 of distilled water and 5 ml of n-butanol & pyridine solution have to be added and shaken vigorously.
4. After centrifugation (4000rpm) for 10 minutes and a supernatant was read at 532nm.
I used to do this protocol by eliminating the adding of distilled water and n-butanol & pyridine solution (The step 3). However, I can get the standard curve with R2 value 0.99. I also performed with 3rd step, but I didn't get the proper standard curve. Please let me clarify that, Is following the original protocol mandatory (with 3rd step) or following this protocol without 3rd step is sufficient.
Thanks in advance for your valuable suggestions.
It is found that vitamin C supplement helps in reducing MDA level in liver disease patient. Further studies suggest that zinc level is reduced in liver patient. So what will be the relation between MDA and Zinc?
Again we know that NO is a lipid peroxidation products. If NO (Nitric oxide) is also increase in liver patient then what will be the relation between zinc and NO?
I have taken data of both assays.If I want to compare antioxidant activity with relation of Lipid peroxidation(By MDA) then which type of calculation/formula/software will used through which I recognized either antioxidant are working well in this biological system or not.
I am doing TBARS assay to measure lipid peroxidation in LPS induced RAW 264.7 cells.I am not getting any result as there is no appearance of pink color in sample after doing assay.I am following this protocol.please find the attached document.
thanks in advance.
I am trying to standardize Lipid per-oxidase assay using TBARS method. At the end of the assay, when I am recording OD of the final pink coloured product, the reading is fluctuating and is undergoing a gradual reduction with time.
My questions are:
What should I do to have a stable OD?
Is it OK to consider the first OD as correct one, shown just after placing the cuvette into the Spectrophotometer?
I have seddlings of sorghum and I could not homogenate them at the detected time of incubation, so I conserved them in the refergerator for 2 days, can I homogenate them and estimate lipid peroxidation and H202?
I want to carry my plant tissue abroad and it is not possible to maintain the samples in -80 centigrade so I need to freeze dry and carry samples. Will it affect the antioxidant enzyme activity?
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-
- Homogenized in Tris-HCl (50 mM, pH-7.4).
- Centrifuged at 12,000g for 10 mins.
- Taken supernatant.
- 50 µl homogenate were mixed with 500 µl of 0.67% TBA, 500 µl 20% trichloroacetic acid.
- The mixtures were incubated in a boiling water bath for 20 min.
- 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.