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

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.