quantifying dna oxidative damage in mcf-7 cells after hydrogen peroxide treatment

Determining the Degree of DNA Oxidative Damage in MCF-7 Cells

DNA oxidative damage is a consequence of an imbalance between the generation of reactive oxygen species (ROS) and the cellular antioxidant defense system1. It primarily involves the oxidation of DNA purine and pyrimidine bases2,3. This oxidative modification of DNA bases not only compromises the integrity of the genome but also encompasses a wide range of pathological issues, including cancer, neurodegenerative diseases, and cardiovascular diseases4,5. The guanine base in DNA has the lowest reduction potential and is the most susceptible to oxidation6. Therefore, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) serves as a primary marker for assessing the extent of DNA oxidative damage7,8. The accurate quantification of 8-oxo-dG has become a critical issue in addressing various aspects of disease occurrence, progression, and the assessment of multifactorial oxidative burden9.
The traditional methods for detecting 8-oxo-dG, such as high-performance liquid chromatography with electrochemical detection (HPLC-ECD), mass spectrometry, and related hyphenated techniques, exhibit high sensitivity and specificity10,11,12. However, these techniques often have complex operational requirements and high costs, which hinder their widespread applicability and practicality in high-throughput sample analysis. With the continuous advancement of science and technology, a variety of new, efficient, and accurate methods have emerged. The application of these new technologies enables us to quantify the level of 8-oxo-dG more accurately and provides more powerful tools for an in-depth study of the association between oxidative stress and disease. For instance, researchers have applied nanopore technology to quantitatively detect and sequence DNA13, identify DNA damage types using a single-click code-sequencing strategy14, develop high-throughput sequencing methods, and create 8-oxoG-based biosensors by integrating biotin-streptavidin with ELISA15. Among them, ELISA, with its recognized advantages in terms of specificity, high-throughput screening, and cost, is one of the ideal solutions for 8-oxo-dG detection. Therefore, it is crucial to develop a high-throughput, highly sensitive, convenient, and rapid method for detecting 8-oxo-dG.
The enzyme-linked immunosorbent assay (ELISA) technique, developed in 197116, has rapidly advanced over the past 50 years and has now become one of the most commonly used detection methods in the fields of biology and medicine17,18,19. ELISA technology exhibits high sensitivity and specificity, possesses a short reaction time, and is easy to use, making it a widely recognized choice for large-scale sample testing and high-throughput analysis20. As a result, ELISA has been widely used for quantitative or semiquantitative analysis of compounds, proteins, antibodies, or molecules within cells21,22,23. For example, it has been utilized in the detection of biomarkers associated with various diseases, drug residues, and biomolecules24. ELISAs can be categorized into four main types based on experimental design and principles25. These methods include direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA26,27. Among these, sandwich ELISA, which utilizes two specific antibodies, one for capturing the target molecule and the other for detection, was chosen for the study in this paper. The experimental principle of sandwich ELISA is as follows: First, a specific antibody is immobilized in the wells of a microplate to capture the analyte of interest. After the standard or sample is added, the target analyte binds to the immobilized antibody. Subsequently, a labeled antibody that recognizes a different epitope on the antigen is added, forming a sandwich structure. Following the removal of unbound antibodies, a substrate is added. Under the catalytic action of the secondary antibody, a color reaction occurs, and the intensity of the color is positively correlated with the concentration of the target analyte in the sample. Finally, the optical density (OD) was measured to determine the concentration of the sample. Sandwich ELISA has the advantages of increased sensitivity and specificity for target samples, which makes it suitable for detecting low concentrations of target analytes and complex samples28. Additionally, the results obtained can be quantified for further analysis. These factors make sandwich ELISA a commonly used detection method in both scientific research and clinical laboratories29.
This study aimed to quantitatively detect 8-oxo-dG in MCF-7 cells to determine the degree of DNA oxidative damage in the cells. This study consists of two main parts: constructing an MCF-7 cell DNA oxidative damage model and detecting 8-oxo-dG using ELISA. First, MCF-7 cells were cultured in vitro and treated with different concentrations of H2O2 for different durations. Cell viability was evaluated using a CCK-8 assay to determine the half-maximal inhibitory concentration (IC50) of H2O2 in MCF-7 cells. Based on the IC50 values, an appropriate H2O2 treatment time and induction concentration were chosen. To extract samples of MCF-7 cells damaged by oxidation, cell samples, and supernatants were obtained and added to enzyme-linked wells previously coated with 8-oxo-dG antibodies. The 8-oxo-dG present in the sample will bind to the antibodies bound to the solid-phase carrier. Then, 8-oxo-dG antibodies labeled with horseradish peroxidase were added. The reaction mixture was incubated at a constant temperature to ensure complete binding of the sample and the antibody. The unbound enzyme was removed by washing, and then the colorimetric substrate was added, which produced a blue color. Under the action of acid, the solution turned yellow. Finally, the OD value of the reaction well samples was measured at 450 nm, and the concentration of 8-oxo-dG in the sample was proportional to the OD value. By generating a standard curve, the concentration of 8-oxo-dG in the sample can be calculated.

Author Spotlight: Quantitative Assessment of 8-oxo-dG in MCF-7 Cells Using ELISA

Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

This research aims to quantitatively measure 8-oxo-dG in MCF-7 cells using ELISA, assessing the impacts of hydrogen peroxide on DNA oxidative damage. Specifically, ELISA steps are detailed in this video for efficiency. Techniques for detecting 8-oxo-dG including high-performance liquid chromatography with electrochemical detection, mass spectrometry, and other sophisticated methodologies have demonstrated efficacy.However, these methods often involve numerous tedious steps and can be quite costly. This protocol addresses the research gap in efficient and sensitive detection of oxidative DNA damage, specifically 8-oxo-dG in cell samples, which is crucial for understanding its implications in disease, aging, and response to environmental stresses. My lab focus on method innovation for DNA damage event detection.This will enable us to conduct in-depth lab science research on DNA damage markers.

Quantifying DNA Oxidative Damage in MCF-7 Cells After Hydrogen Peroxide Treatment

quantifying-dna-oxidative-damage-mcf-7-cells-after-hydrogen-peroxide

Research

JoVE Journal

Biochemistry

88 visualizações.  Suzhou Vocational Health College. Depois de gerar a cultura de células MCF-7 induzida por peróxido de hidrogênio, descarte o sobrenadante da placa de cultura. Adicione um mililitro de PBS e agite suavemente o prato antes de descartar o PBS. Agora adicione um mililitro de tripsina para lavar as células, sacuda o prato e espere um minuto, depois descarte a tripsina.Bata suavemente no prato, observe o movimento da célula na folha para confirmar o descolamento. Em seguida, adicione quatro mililitros de meio de cultura...