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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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+proteomic+strategies+for+identifying+tumour-associated+antigens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+das-ELISA+for+gyrovirus+homsa1+prevalence+survey+in+chickens+and+wild+birds+in+China.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hogg1+expression+level+on+oxidative+DNA+damage+among+workers+exposed+to+nickel+in+stainless+steel+production+environment.">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.
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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.

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			<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>CO2 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>H2O2(3%)</td>
			<td>Jiangxi Caoshanhu Disinfection Co.,Ltd.</td>
			<td>1028348</td>
			<td></td>
		</tr>
		<tr>
			<td>high-speed centrifuge</td>
			<td>Thermo</td>
			<td>&#160;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>
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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 sugge...

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

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

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 ...

quantifying-level-8-oxo-dg-using-elisa-assay-to-evaluate-oxidative

Research

JoVE Journal

Biochemistry

760 Views.  Suzhou Vocational Health College. This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.

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

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-&#946; peptide variants with their respective intrinsic aggregation propensities.

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-&#946; peptide.

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+- and O2- sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...