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

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....

Materials

List of materials used in this article

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