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Cell Meter™ Intracellular Colorimetric Lipid Peroxidation (MDA) Assay Kit

MDA dose response was measured with Amplite™ Colorimetric Lipid Peroxidation (MDA) Quantitation Kit on a 96-well clear bottom microplate using a SpectraMax microplate reader (Molecular Devices).
MDA dose response was measured with Amplite™ Colorimetric Lipid Peroxidation (MDA) Quantitation Kit on a 96-well clear bottom microplate using a SpectraMax microplate reader (Molecular Devices).
Signal Comparison of MDA, Formaldehyde, and Glyceraldehyde.
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Catalog Number15991
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Telephone1-408-733-1055
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Lipid peroxidation is characterized by the oxidative degradation of unsaturated fatty acids, phospholipids, glycolipids, cholesterol esters and cholesterol. Malondialdehyde (MDA) is one of the most commonly used biomarkers for lipid peroxidation. Measurement of MDA has historically relied on a reaction with thiobarbituric acid (TBA) to generate a product that can be measured colorimetrically or fluorimetrically. However, TBA assay has quite a few limitations. (1). The reaction is not specific to MDA. (2). TBA-MDA reaction need be run under acidic conditions. (3). The assays need be run under high temperature, commonly at 90-100 ºC. The commercial TBA-based MDA assays are extremely tedious to run due to these limitations. This Cell Meter™ Colorimetric Lipid Peroxidation (MDA) Quantitation Kit offers the most rapid and convenient method to measure MDA without the TBARS heating steps. MDA Blue™ reacts with MDA to generate a blue color product which is measured at 695 nm with absorbance microplate reader. This assay is very fast and specific to MDA with little interference from other aldehydes.

Platform


Absorbance microplate reader

Recommended plateClear bottom

Components


Component A: MDA Blue™1 vial
Component B: Dilution Buffer1 bottle (10 mL)
Component C: MDA Standard1 vial (lyophilized)
Component D: Reaction Solution1 bottle (10 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare and add MDA standards and/or test samples (50 µL)
  2. Prepare and add MDA Blue™ (10 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. Add reaction solution (40 µL)
  5. Monitor OD increase at 695 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

MDA standard stock solution (100 mM):
Add 100 µL of ddH2O into MDA Standard (Component C) and mix them well. Note: The unused MDA stock solution should be stored at -20°C in single use aliquots.

PREPARATION OF STANDARD SOLUTION

MDA standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15991

Add 4 μL of MDA standard (100 mM) into 996 μL of Dilution Buffer (Component B) to get MDA standard solution (400 μM). Then perform 1:2 serial dilutions in dilution buffer to get 200, 100, 50, 25, 12.5, 6.25 and 0 μM serially diluted MDA standards.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of MDA standards and test samples in a clear bottom 96-well microplate. MDA = MDA standard (MDA1-MDA7= 400 to 6.25 µM); BL = blank control; TS = test sample.

BL BL TS TS
MDA1 MDA1 ... ...
MDA2 MDA2 ... ...
MDA3 MDA3    
MDA4 MDA4    
MDA5 MDA5    
MDA6 MDA6    
MDA7 MDA7    

Table 2. Reagent composition for each well.

Well Volume Reagent
MDA1-MDA7 50 µL Serial Dilution (400 to 6.25 µM)
BL 50 µL Dilution Buffer (Component B)
TS 50 µL Test Sample

MDA assay

  1. Add 50 µL of MDA standard, blank control, and test samples to clear bottom 96-well microplate (As shown in Table 1 and Table 2).

  2. Add 10 µL/well of MDA Blue™ (Component A) into each well of MDA standard, blank control and test samples. Note: For a 384-well plate, add 25 µL of sample, 5 µL of MDA Blue™ solution into each well. Note: Please aliquot Component A into single use size and store unused at -20oC and avoid light.

  3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.

  4. Add 40 µL of Reaction Solution (Component D) to make the total assay volume of 100 µL/well. Note: For a 384-well plate, add 20 µL of Reaction Solution (Component D) to make the total assay volume of 50 µL/well.

  5. Monitor absorbance increase with an absorbance plate reader with path-check correction at OD of 695~700 nm.

References


View all 21 references: Citation Explorer
Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges.
Authors: Tsikas D., undefined
Journal: Anal Biochem (2016)
Formation of Malondialdehyde, 4-Hydroxynonenal, and 4-Hydroxyhexenal during in Vitro Digestion of Cooked Beef, Pork, Chicken, and Salmon
Authors: Steppeler C, Haugen JE, Rodbotten R, Kirkhus B.
Journal: J Agric Food Chem (2016): 487
Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15
Authors: Tsikas D, Rothmann S, Schneider JY, Suchy MT, Trettin A, Modun D, Stuke N, Maassen N, Frolich JC.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2016): 95
Plasma malondialdehyde as biomarker of lipid peroxidation: effects of acute exercise
Authors: Spirl, undefined and eli AL, Deminice R, Jordao AA.
Journal: Int J Sports Med (2014): 14
A specific, accurate, and sensitive measure of total plasma malondialdehyde by HPLC
Authors: Moselhy HF, Reid RG, Yousef S, Boyle SP.
Journal: J Lipid Res (2013): 852
Antioxidant properties of Krebs cycle intermediates against malonate pro-oxidant activity in vitro: a comparative study using the colorimetric method and HPLC analysis to determine malondialdehyde in rat brain homogenates
Authors: Puntel RL, Roos DH, Grotto D, Garcia SC, Nogueira CW, Rocha JB.
Journal: Life Sci (2007): 51
Malondialdehyde as biomarker of oxidative damage to lipids caused by smoking
Authors: Lykkesfeldt J., undefined
Journal: Clin Chim Acta (2007): 50
Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection
Authors: Grotto D, Santa Maria LD, Boeira S, Valentini J, Charao MF, Moro AM, Nascimento PC, Pomblum VJ, Garcia SC.
Journal: J Pharm Biomed Anal (2007): 619
Glaucoma and oxidative stress. Determination of malondialdehyde--a product of lipid peroxidation
Authors: Faschinger C, Schmut O, Wachswender C, Mossbock G.
Journal: Ophthalmologe (2006): 953
Short-term oral ingestion of Ginkgo biloba extract (EGb 761) reduces malondialdehyde levels in washed platelets of type 2 diabetic subjects
Authors: Kudolo GB, Delaney D, Blodgett J.
Journal: Diabetes Res Clin Pract (2005): 29