Name: Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells (Video) | JoVE Journal
Uploaded: 2024-05-24T14:40:00.000+00:00
Duration: 5 min 13 s
Description: Learn to quantify 8-oxo-dG levels using ELISA assay to evaluate oxidative DNA damage in MCF-7 cells. This efficient protocol measures hydrogen peroxide effects for disease and aging research.

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5 minutes

This work was supported by the Jiangsu Higher Education Institution Innovative Research Team for Science and Technology (2021), Program of Jiangsu Vocational College Engineering Technology Research Center (2023), Key Technology Programme of Suzhou People&#39;s Livelihood Technology Projects (SKY2021029), Open Project of Jiangsu Biobank of Clinical Resources (TC2021B009), Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (GZK12023013), Programs of the Suzhou Vocational Health College (SZWZYTD202201), and Qing-Lan Project of Jiangsu Province in China (2021, 2022).

1. Construction of an H2O2 -induced DNA oxidative damage model in MCF-7 cells
<ol>
	<li>MCF-7 cell recovery
	<ol>
		<li>Transfer the cell culture cryogenic tube, which contains 3.5 x 106 MCF-7 cells and is stored in a -80&#176;C refrigerator, rapidly to a foam box containing liquid nitrogen. Retrieve the tube with forceps and place it in a 37 &#176;Cconstant temperature water bath for approximately 1 min to thaw the preserved cells. 
		NOTE: During the thawing process in a water bath, shake the cells intermittently to ensure uniform heating of the cryovial.</li>
		<li>Place the cryovial in a centrifuge and centrifuge at room temperature at 150 x g for 5 min.</li>
		<li>Use a pipette to discard the supernatant from the cryogenic storage tube and add 1 mL of complete culture medium. Mix well and transfer to a 10 mm culture dish, supplement an additional 7 mL of complete culture medium.</li>
		<li>Shake the culture dish in a crisscross pattern to ensure uniform mixing, and culture in a CO2 incubator at 37 &#176;Cwith 5% CO2. 
		NOTE: The complete culture medium consists of DMEM culture medium supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin.</li>
	</ol>
	</li>
	<li>Cell viability assessment
	<ol>
		<li>Select MCF-7 cells in the logarithmic growth phase with good cellular status for the experiment. During the logarithmic growth phase, cells typically exhibit a shorter division cycle and a high frequency of cell division.
		<ol>
			<li>Observe the morphology and growth rate of MCF cells using an electron microscope. When the cells present a uniform and densely packed monolayer morphology, and cell proliferation is evident, it can be determined that the cells are essentially in the logarithmic growth phase.</li>
		</ol>
		</li>
		<li>Cell washing: Use a pipette to remove the culture medium from the culture dish, add 1 mL of PBS buffer to wash the cells, then discard the PBS.</li>
		<li>Cell detachment: Add 1 mL of trypsin to wash the cells, wait for about 1 min for trypsin to completely detach the cells from the culture dish. Gently tap the culture dish; observation of cell movement 
		in sheets indicates thorough cell detachment.</li>
		<li>Termination of cell detachment and cell counting: Add 8 mL of complete culture medium to terminate the detachment, gently pipette cells, and determine the cell suspension concentration using a cell counting device. 
		NOTE: Ensure cell suspension is homogenous before counting by gently pipetting up and down.</li>
		<li>Cell seeding: Adjust the diluted cell suspension to achieve a cell density of 5,000 cells per 100 &#181;L. Inoculate the cell suspension into 21 wells of a 96-well plate using a pipette gun, dispensing 100 &#181;L into each well. Add 100 &#181;L of PBS buffer to the surrounding wells of the seeded wells to prevent excessive evaporation of the culture medium.</li>
		<li>Preparation of H2O2-containing medium: Choose 9 wells on the 12-well cell culture plate and assign labels based on the gradient of H2O2 concentrations (0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0 mM). Add 720 &#181;L of complete medium to the wells labeled as 2.0 mM and 1.5 mM and add 360 &#181;L of complete medium to the remaining wells.
		<ol>
			<li>Using a pipette, dispense 1.63 &#181;L and 1.22 &#181;L of 3% H2O2 reagent into the wells labeled as 2.0 mM and 1.5 mM, respectively. Mix the solution thoroughly. Using a pipette, transfer 360 &#181;L of 2.0 mM H2O2 into the well labeled 1.0 mM and mix; then, transfer 360 &#181;L of 1.0 mM H2O2 into the well marked 0.5 mM and mix; and finally transfer 360 &#181;L of 0.5 mM H2O2 into the well marked 0.25 mM. Pipette 360 &#181;L of 1.5 mM H2O2 into the well labeled 0.75 mM to dilute and achieve the desired concentration. (Figure 1). 
			&#8203;NOTE: Ensure that the H2O2 stock solution in the pipette is completely transferred into the wells to avoid any residual solution. Ensure thorough mixing of the solutions before proceeding with the dilution.</li>
		</ol>
		</li>
		<li>Incubation: After approximately 24 h of cell seeding, use a pipette to remove the culture medium from the 96-well plate once cells have adhered to the surfaces. As per the concentration gradient, add the respective H2O2-containing medium to each well. Return the plate to the incubator for 12 h.</li>
		<li>Addition of CCK-8 reagent: Following the designated 12 h treatment period, use a pipette to remove the culture medium from the 96-well plate, add 100 &#181;L of complete culture medium and 10 &#181;L of CCK-8 reagent to each well, and include three blank control wells. Incubate the plate at 37 &#176;Cfor 2 h. 
		NOTE: During the process of adding samples, avoid the formation of bubbles to prevent any interference with the OD value reading.</li>
		<li>Absorbance measurement: Take out the 96-well plate from the incubator, measure the absorbance at a wavelength of 450 nm using a microplate reader, and record the results.</li>
		<li>According to the IC50 calculation formula, 
		inhibition rate (%) = (OD of drug experimental group-average OD of drug control hole)/ (average OD of cell control hole-average OD of drug control hole) x 100% 
		Calculate results as percentage of absorbance in control cultures.</li>
		<li>Import the processed experimental data into a statistical analysis software, select the analysis type Nonlinear Regression, construct the four-parameter logistic model, and proceed to click the Fit Curve button for curve fitting. Following the fitting process, the software will automatically compute the IC50 value.</li>
	</ol>
	</li>
	<li>H2O2-induced MCF-7 cell oxidation experiment
	<ol>
		<li>Retrieve MCF-7 cells in the logarithmic growth phase with good cell status for experimentation.</li>
		<li>Wash, perform cell detachment, terminate the detachment, and count the cells (refer to step 1.2 for specific methods).</li>
		<li>Cell seeding: Adjust the density of the cell suspension to 1 x 106 cells per dish, with 4 mL of the suspension per 6 cm diameter dish.</li>
		<li>Label two 5 mL microcentrifuge tubes as 0.25 mM and 0.75 mM 3% H2O2. Add 4 mL of complete medium to each tube, then add 1.13 &#181;L and 3.40 &#181;L of 3% H2O2, respectively. Gently shake the tubes to ensure thorough mixing. Replace the culture medium in the three dishes with a complete medium containing H2O2 concentrations of 0, 0.25, and 0.75 mM.</li>
		<li>Incubate the cultures: Return the dishes to the incubator for 12 h.</li>
	</ol>
	</li>
</ol>
2. Preparation of cellular samples and ELISA reagents preparation
<ol>
	<li>Collection of cell extract samples
	<ol>
		<li>Sample collection: Discard MCF-7 cell culture supernatant, rinse, perform cell detachment, terminate the detachment, and collect cells. Transfer the cell suspension to a 15 mL centrifuge tube.</li>
		<li>Centrifugation: Centrifuge at 800 x g for 5 min to collect the cell pellet. Add 200 &#181;L of PBS to resuspend each cell sample and disrupt cells by repeated freeze-thaw cycles. Centrifuge the cell lysate at 1,500 x g for 10 min and use a pipette to collect the supernatant for analysis. 
		NOTE: When the cell amount is low, reduce the volume of PBS used for resuspension. Samples can be stored at 4 &#176;C for up to 1 week if immediate analysis is not possible. For long-term storage, aliquot the samples based on single-use quantities and store them at -80 &#176;C to avoid repeated freeze-thaw cycles.</li>
	</ol>
	</li>
	<li>Preparation of ELISA reagents
	<ol>
		<li>Thawing of reagents: Allow the ELISA reagents, including seal films, precoated plates, standards, sample diluent, detection antibody-HRP, chromogen substrate, stop solution, concentrated wash buffer (20x), and the test samples to reach room temperature (18-25 &#176;C) for 20 min before starting the assay. Pre-open the ELISA reader 15 min before use.</li>
		<li>Preparation of wash buffer: Calculate the required volume of diluted wash buffer, and then dilute 20x concentrated wash buffer with distilled or deionized water into 1x working solution. Return any unused concentrated wash buffer to the 4 &#176;C refrigerator. 
		NOTE: The wash buffer should be prepared fresh before use. If crystals are present in the concentrated wash buffer stored in the refrigerator, allow it to reach room temperature and gently shake it until the crystals dissolve completely before utilization.</li>
		<li>Preparation of standard
		<ol>
			<li>Prepare working solutions of standard samples with concentrations of 1.25, 2.5, 5, 10, 20, and 40 ng/mL, respectively. To ensure the accuracy of the experimental results, carefully mix the standard samples up and down before use to avoid the formation of bubbles during the dissolution process.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. ELISA experiment
<ol>
	<li>Plate coating: Design the total number of wells for standards, samples, and blank/controls in advance and take out the required plate strips. Add 50 &#181;L of standard working solution and detection samples with different concentrations to the reaction wells. Perform standards in triplicates and refrain from adding any samples to the blank/control wells25. 
	NOTE: The experimental steps of the ELISA assay are shown schematically in Figure 2. If the sample concentration exceeds the detection range, the sample should be diluted with sample diluent before sampling. When adding samples, add them to the bottom of the plate, avoiding touching the well walls, gently shake to mix, and avoid the formation of bubbles. To ensure the accuracy of the experiment, each sample addition should be completed within 10 min.</li>
	<li>Incubation: Except for the blank wells, add 100 &#181;L of horseradish peroxidase (HRP)-conjugated detection antibody to each reaction well, seal the wells with a plate seal, and incubate at 37 &#176;C for 60 min in a constant temperature incubator.</li>
	<li>Washing: Discard the liquid, shake off the liquid in the plate wells, pat dry on absorbent paper, add 350 &#181;L of wash buffer to each reaction well, let it stand for 1-2 min, then discard the wash buffer. Repeat the washing process 5x. 
	NOTE: Thorough washing is crucial because it directly affects the accuracy of the experimental results. Ensure that the wash buffer is completely shaken off after each washing. Carefully wipe off any residue on the bottom of the plate after washing to avoid affecting the absorbance measurement.</li>
	<li>Color reaction: Add 50 &#181;L of substrate A and 50 &#181;L of substrate B to each reaction well, seal the plate and incubate at 37 &#176;C in the dark for 15 min. 
	NOTE: After adding the color reagent, observe the color change in the reaction wells promptly. If the color is too dark, add the stop solution to terminate the reaction earlier.</li>
	<li>Terminate the reaction and measure the absorbance: Add 50 &#181;L of stop solution to each reaction well, and immediately measure the OD value at 450 nm wavelength with an ELISA reader (within 15 min).</li>
	<li>Data analysis: Import the processed experimental data into the statistical analysis software, select the analysis type Linear Regression. Construct a calibration curve by plotting the concentration of the standards on the X-axis against the optical density (OD) values on the Y-axis. Utilize the established calibration curve to ascertain the concentration of 8-oxo-dG present in the samples. Construct a Logistic mathematical model and proceed to click the Fit Curve button for curve fitting. Following the fitting process, the software automatically calculates the standard curve and P-value. 
	NOTE: The standard curve cannot be reused and needs to be determined anew for each experiment.</li>
</ol>

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

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<li>Larsson, P., Parris, T. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+cell+viability+assays+for+drug+sensitivity+screens%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Optimization of cell viability assays for drug sensitivity screens.</a> Methods Mol Biol. 2644, 287-302 (2023).</li>
<li>Gunawardana, C. G., Diamandis, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+proteomic+strategies+for+identifying+tumour-associated+antigens%5BTitle%5D)+AND+%22Cancer+Lett%22%5BJournal%5D)">High throughput proteomic strategies for identifying tumour-associated antigens.</a> Cancer Lett. 249 (1), 110-119 (2007).</li>
<li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+das-ELISA+for+gyrovirus+homsa1+prevalence+survey+in+chickens+and+wild+birds+in+China%5BTitle%5D)+AND+%22Vet+Sci%22%5BJournal%5D)">Development of a das-ELISA for gyrovirus homsa1 prevalence survey in chickens and wild birds in China.</a> Vet Sci. 10 (5), 312(2023).</li>
<li>Li, Y., Sun, J., Shang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+hogg1+expression+level+on+oxidative+DNA+damage+among+workers+exposed+to+nickel+in+stainless+steel+production+environment%5BTitle%5D)+AND+%22Zhonghua+Lao+Dong+Wei+Sheng+Zhi+Ye+Bing+Za+Zhi%22%5BJournal%5D)">Effect of hogg1 expression level on oxidative DNA damage among workers exposed to nickel in stainless steel production environment.</a> Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 32 (8), 578-581 (2014).</li>
</ol>

The authors have no conflicts of interest to disclose.

The development of ELISA methods holds great importance for both existing and new DNA damage detection methodologies. In comparison to traditional HPLC and mass spectrometry techniques, this approach not only is user-friendly but also exhibits high sensitivity and meets the demands of high-throughput screening30. This enables the monitoring of 8-oxo-dG in large-scale disease screening studies, facilitating a deeper understanding of the correlation between this biomarker and various diseases.
During experimental procedures, several critical steps are essential to ensure the reliability and reproducibility of the results. Initially, the experiment proficiently assessed the sensitivity of MCF-7 cells to H2O2, with an IC50 value of 0.7655 mM at 12 h, highlighting the tolerance level of MCF-7 cells. This outcome is of great scientific significance for understanding the antioxidant defense mechanisms of MCF-7 cells and their survival under oxidative stress while providing an important reference indicator for future screening of antioxidant drugs or related treatment mechanisms31. Furthermore, the selection and pairing of 8-oxo-dG antibodies is vital for ELISA detection and directly affects the specificity and sensitivity of the ELISA detection system. In addition, optimizing the signal amplification strategy and sample processing steps are important factors that affect the accuracy of detection. We found that using a stable substrate reaction system can reduce background errors and increase the sensitivity range of detection.
During the experimental process, technical issues such as unstable antibodies, signal fluctuations, and cross-reactivity of reactants may occur. To address these issues, we adopted a more stable experimental protocol, which included the use of stable antibody solvents to improve antibody stability and the optimization and adjustment of experimental conditions. Additionally, to address the error caused by signal fluctuations, we standardized the experiment using standard 8-oxo-dG samples to ensure consistency of the results.
Regarding the limitations of the experimental method, although ELISA provides a high-throughput approach, it may still be limited by the specific antibody coverage and inevitable substrate cross-reactivity32. Therefore, additional sample purification or preprocessing steps may be required for complex samples. Moreover, the detection of samples with lower concentrations may necessitate further optimization of antibody activity to meet the research requirements.
In conclusion, the ELISA detection method for 8-oxo-dG has important implications for specific research areas. It has potential application value in environmental monitoring, disease risk assessment, and early diagnosis and provides a new detection approach for 8-oxo-dG in agriculture, food safety, and drug screening33. For example, in environmental biomonitoring, the detection of DNA oxidative damage stress caused by environmental toxins in organisms can help identify potential risks in a timely manner34. In the future, with further optimization of detection technology and its application in multiple disciplines, this method is expected to play a more critical role in life science research.

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.

<table><tbody><tr><td>0.25% Trypsin-EDTA(1x)</td><td>Gibco</td><td>25200-072</td><td></td></tr><tr><td>Cell Counting Kit-8</td><td>Dojindo</td><td>CK04</td><td></td></tr><tr><td>Cell Counting Plate</td><td>QiuJing</td><td>XB-K-25</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo</td><td>51032872</td><td></td></tr><tr><td>DMEM basic(1X)</td><td>Gibco</td><td>C11995500BT</td><td></td></tr><tr><td>FBS</td><td>PAN</td><td>ST30-3302</td><td></td></tr><tr><td>GraphPad Prism X9</td><td>GraphPad Software</td><td></td><td>statistical analysis software</td></tr><tr><td>high-speed centrifuge</td><td>Thermo</td><td>&amp;nbsp;9AQ2861</td><td></td></tr><tr><td>Human 8-oxo-dG ELISA Kit</td><td>Zcibio</td><td>ZC-55410</td><td></td></tr><tr><td>L-1000XLS+ Pipettes</td><td>Rainin</td><td>17014382</td><td></td></tr><tr><td>L-20XLS+ Pipettes</td><td>Rainin</td><td>17014392</td><td></td></tr><tr><td>liquid nitrogen tank</td><td>Mvecryoge</td><td>YDS-175-216</td><td></td></tr><tr><td>MCF-7 CELL</td><td>BNCC</td><td>BNCC100137</td><td></td></tr><tr><td>Multiskan FC microplate photometer</td><td>Thermo</td><td>1410101</td><td></td></tr><tr><td>PBS</td><td>Solarbio</td><td>P1020</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100X</td><td>Beyotime</td><td>C0222</td><td></td></tr><tr><td>Trinocular live cell microscope</td><td>Motic</td><td>1.1001E+12</td><td></td></tr><tr><td>Ultra-low temperature freezer</td><td>Haire</td><td>V118574</td><td></td></tr></tbody></table>

quantifying the level of 8-oxo-dg using elisa assay to evaluate oxidative dna damage in mcf-7 cells

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

8-Oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression and serves as a pivotal factor in stimulating neurodegenerative disorders, cancer, and aging. Therefore, precise quantification of 8-oxoG has clinical significance in the investigation of DNA damage detection methodologies. However, at present, the existing approaches for 8-oxoG detection pose challenges in terms of convenience, expediency, affordability, and heightened sensitivity. We employed the sandwich enzyme-linked immunosorbent assay (ELISA) technique, a highly efficient and swift colorimetric method, to detect variations in 8-oxo-dG content in MCF-7 cell samples stimulated with different concentrations of hydrogen peroxide (H2O2). We determined the concentration of H2O2 that induced oxidative damage in MCF-7 cells by detecting its IC50 value in MCF-7 cells. Subsequently, we treated MCF-7 cells with 0, 0.25, and 0.75 mM H2O2 for 12 h and extracted 8-oxo-dG from the cells. Finally, the samples were subjected to ELISA. Following a series of steps, including plate spreading, washing, incubation, color development, termination of the reaction, and data collection, we successfully detected changes in the 8-oxo-dG content in MCF-7 cells induced by H2O2. Through such endeavors, we aim to establish a method to evaluate the degree of DNA oxidative damage within cell samples and, in doing so, advance the development of more expedient and convenient approaches for DNA damage detection. This endeavor is poised to make a meaningful contribution to the exploration of associative analyses between DNA oxidative damage and various domains, including clinical research on diseases and the detection of toxic substances.

As illustrated in Figure 3, the X-axis denotes the concentration of H2O2 applied to MCF-7 cells, while the Y-axis indicates cell viability. Treatment with 0.75 mM for 12 h reduced the viability of MCF-7 cells to 67%. Concomitant with the increase in concentration, there was a significant decrease in the viability of MCF-7 cells, particularly at a concentration of 1.5 mM, where the viability decreased to below 3% (Table 1). The experimental results suggested an IC50 of 0.7655 mM for the MCF-7 cells.
In this series of experiments, we employed an enhanced ELISA technique for the quantification of 8-oxo-dG levels. Through data analysis, the regression equation of the standard curve is:
Y = 23.66 × X + 0.07038.
As depicted in Figure 4A, the calibration curve demonstrated high linearity (R²=0.978), and Figure 4B shows that the relationships between the measured concentrations of 8-oxo-dG in the samples and their corresponding added amounts were strongly correlated, indicating good precision (the coefficient of variation was less than 5%). Moreover, the consistency of the obtained results across multiple replicates indicates the reliability and reproducibility of the experimental method.
In summary, the experimental data indicate that our optimized ELISA method is capable of successfully detecting fluctuations in 8-oxo-dG levels under well-controlled conditions. This has significant implications for future applications of this technique in a wider range of sample types to monitor DNA damage caused by oxidative stress. Despite certain limitations, the method's prospects for application appear promising upon optimization of the experimental conditions.
<img alt="Serial dilution diagram; H2O2 concentration preparation, stepwise reduction, lab technique." class="xfigimg" src="/files/ftp_upload/66888/66888fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Preparing H2O2 dilutions. The schematic diagram delineates the method for preparing hydrogen peroxide (H2O2) dilutions, involving the meticulous combination of solutions and the precise transfer of varied H2O2 concentrations into tubes labeled as 1.0 mM, 0.5 mM, and 0.25 mM correspondingly. The targeted concentration is attained by transferring 1.5 mM H2O2 into the tube designated as 0.75 mM. <a href="https://www.jove.com/files/ftp_upload/66888/66888fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="ELISA process diagram; antibody detection, antigen labeled with HRP, substrate reaction setup." class="xfigimg" src="/files/ftp_upload/66888/66888fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Enzyme-linked immunosorbent Assay (ELISA) for antigen detection. This schematic illustration depicts the sequential steps of an Enzyme-Linked Immunosorbent Assay (ELISA) for antigen detection: Sample addition and blank preparation; HRP-labeled antigen incubation; washing and plate preparation; substrate addition and incubation; reaction termination and optical density (OD) measurement. <a href="https://www.jove.com/files/ftp_upload/66888/66888fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell viability vs. H₂O₂ concentration graph; IC50=0.7655 mM; dose-response curve analysis." class="xfigimg" src="/files/ftp_upload/66888/66888fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: H2O2 inhibited MCF-7 cell proliferation. Cell viability was evaluated by CCK-8 assay treated with various concentrations of H2O2 (0, 0.25, 0.50, 0.75, 1.0, 1.5, or 2.0 mM) for 12 h and IC50 was calculated for the indicated condition. <a href="https://www.jove.com/files/ftp_upload/66888/66888fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Standard curve and bar graph; 8-oxo-dG quantification vs. OD value, H2O2 concentrations analysis." class="xfigimg" src="/files/ftp_upload/66888/66888fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Quantitative analysis of 8-oxo-dG in MCF-7 cells following H2O2 treatment. (A) ELISA standard curve for 8-oxo-dG. Depicts the relationship between known concentrations of 8-oxo-dG (1.25, 2.5, 5, 10, 20, and 40 ng/mL) and their respective absorbance values. The regression equation of the standard curve is Y = 23.66 × X + 0.07038. (n=3, R2=0.978). (B) 8-oxo-dG concentration in H2O2-treated MCF-7 cells. The graph displays the levels of 8-oxo-dG in MCF-7 cells subjected to different concentrations of H2O2 (0, 0.25, and 0.75 mM). Each data point represents the mean ± SE (standard error) from three independent experiments (n=3), illustrating the impact of oxidative stress on DNA damage. <a href="https://www.jove.com/files/ftp_upload/66888/66888fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2 concentration, mM</td>
			<td>0</td>
			<td>0.25</td>
			<td>0.5</td>
			<td>0.75</td>
			<td>1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>MCF-7 cell viability, %</td>
			<td>100</td>
			<td>82.8</td>
			<td>81.37</td>
			<td>61.07</td>
			<td>13.4</td>
			<td>2.77</td>
		</tr>
	</tbody>
</table>
Table 1: Cell viability of MCF-7 cells treated with H2O2.

This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.

8-Oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly ...

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Learn to quantify 8-oxo-dG levels using ELISA assay to evaluate oxidative DNA damage in MCF-7 cells. This efficient protocol measures hydrogen peroxide effects for disease and aging research.

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

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