Thiobarbituric Acid-Reactive Substances Assay to Assess Oxidative Stress in Human Serum

Published: April 30, 2023

Abstract

Source: Borges, C. R. et al., Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay. J. Vis. Exp. (2020)

This video demonstrates the thiobarbituric acid reactive substances (TBARS) assay to assess oxidative stress in biological samples.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board

1. TBARS assay

NOTE: Once the TBARS assay is started, it should be finished without stopping.

  1. Take as many samples and standards centrifuged at 1500 x g for 10 min at 4 °C. After centrifugation, keep the glass tubes containing the samples and standards at RT. glass tubes as needed for the number of samples to be analyzed and label them with the names of the samples. Then, add 100 μL of each prepared samples (HepG2 cell lysates, low-density lipoproteins, human serum) to each glass tube.
  2. Add 200 μL of 8.1% SDS to each sample and standard and gently swirl the glass tube in a circular motion to mix the sample.
  3. Add 1.5 mL of the 3.5 M sodium acetate buffer (pH = 4) to each sample and standard.
  4. Add 1.5 mL of the 3.5 M sodium acetate buffer (pH = 4) to each sample and standard.
  5. Bring the final volume to 4 mL for each sample and standard by adding 700 μL of DI water.
  6. Tightly cap each glass tube and incubate in a heating block set to 95 °C for 1 h. Cover the glass tubes with aluminum foil to prevent condensation at the tops of the tubes.
  7. Remove the glass tubes from the heating block and incubate on ice for 30 min.
  8. Centrifuge samples and standards at 1500 x g for 10 min at 4 °C. After centrifugation, keep the glass tubes containing the samples and standards at RT.
    NOTE: Keeping the samples on ice or at 4 °C will cause the entire sample or standard to precipitate.
  9. Immediately after centrifugation, aliquot 150 μL of supernatant from each tube and place into a separate well of a 96-well plate.
  10. Remove any bubbles from each well using a pipette tip.
    NOTE: The presence of bubbles will yield inconsistent absorbance readings, leading to high assay imprecision.
  11. Read absorbances at 532 nm. Subtract the average absorbance reading of the blank samples from all other absorbance readings.
  12. Create a standard curve by plotting the blank-subtracted absorbance readings at 532 nm vs. the known concentration of each standard. Fit the data points using linear regression. Calculate unknown sample concentrations by using the equation of the linear regression line obtained from the standard curve.

Disclosures

The authors have nothing to disclose.

Materials

1x Sterile PBS pH 7.4 1 L VWR, PA 101642–262 Cell lysis reagent
50 mL self-standing centrifuge tube Corning, NY CLS430897 General material
96 well plate, Non-Treated, clear, with lid, Non-sterile Thermo Fisher Scientific, MA 280895 To measure absorbance
Amicon Ultra-0.5 100 kD centrifugal spin filter device Fisher Scientific, NH UFC510024 LDL purification
Caps for glass tubes Thermo Fisher Scientific, MA 14-930-15D For TBARS assay
Copper II Chloride SIGMA, MO 222011-250G To induce oxidation
Culture tubes, Disposable, with Screw-Cap Finish, Borosilicate Glass (13 x 100 mm) VWR, PA 53283-800 For TBARS assay
Eagle's Minimum Essential Medium (EMEM) ATCC, VA HB-8065 HepG2 cell media
Eppendorf Safe-Lock Tubes, 1.5 mL eppendorf, NY 22363204 General material
Eppendorf Safe-Lock Tubes, 2.0 mL Genesee Sceitific, CA 22363352 General material
Fetal Bovine Serum US Source Omega Scientific, CA FB-11 For cell culture
Glacial Acetic Acid SIGMA, MO 27225-1L-R TBARS Reagent
Halt Protease Inhibitor Cocktail (100x) Thermo Scientific, MA 87786 Cell lysis reagent
HEPES SIGMA, MO H3375-250G LDL solvent
Hydrochloric acid (HCl) Fisher Scientific, NH A144-212 Cell lysis reagent
Legend Micro 17 Centrifuge Thermo Scientific, MA 75002431 General material
Low Density Lipoprotein, Human Plasma Athens Research & Technology, GA 12-16-120412 Biological matrix prototype
Magnetic Stir Bars, Octagon 6-Assortment VWR, PA 58948-025 General material
Malondialdehyde bis (dimethyl acetal) SIGMA, MO 8207560250 TBARS Standard
Multiskan Go Microplate Spectrophotometer Fisher Scientific, NH 51119200 To measure absorbance
NP-40 EMD Millipore Corp, MA 492016-100ML Cell lysis reagent
Sodium Chloride SIGMA, MO S7653-1KG Cell lysis reagent
Sodium dodecyl sulfate (SDS) SIGMA, MO 436143-100G TBARS Reagent
Sodium hydroxide SIGMA, MO 367176-2.5KG TBARS Reagent
SpeedVac Concentrator Thermo Scientific, MA SC250EXP For concentrating cell lysates
Thiobarbituric Acid SIGMA, MO T5500-100G TBARS Reagent
TRIS base Fluka, GA 93362 Cell lysis reagent

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Cite This Article
Thiobarbituric Acid-Reactive Substances Assay to Assess Oxidative Stress in Human Serum. J. Vis. Exp. (Pending Publication), e21326, doi: (2023).

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