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

Overview

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.

Materials

Name	Company	Catalog Number	Comments
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