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. Take as many samples and standards centrifuged at 1500 x g for 10 min at 4 °C. After centrifugation, keep the g…
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).