这项工作得到了江苏省高等学校科技创新研究团队（2021）、江苏省职业学院工程技术研究中心项目（2023）、苏州市民生科技项目关键技术项目（SKY2021029）、江苏省临床资源生物样本库开放项目（TC2021B009）、放射医学与防护国家重点实验室项目、 苏州大学 （GZK12023013）、苏州职业健康学院 （SZWZYTD202201） 项目和中国江苏省青兰项目（2021 年、2022 年）。

1. 在MCF-7 细胞中构建 H2O2 诱导的 DNA 氧化损伤模型
<ol><li>MCF-7 细胞回收<ol><li>将含有 3.5 x 106 个 MCF-7 细胞并储存在 -80°C 冰箱中的细胞培养低温管快速转移到含有液氮的泡沫盒中。用镊子取出试管，并将其置于 37 °C 恒温水浴中约 1 分钟以解冻保存的细胞。 注:在水浴中解冻过程中，间歇性摇动细胞，以确保冻存管均匀加热。</li>
		<li>将冷冻管放入离心机中，并在室温下以 150 x g 离心 5 分钟。</li>
		<li>使用移液器从低温储存管中弃去上清液，并加入 1 mL 完全培养基。充分混合并转移至 10 mm 培养皿中，再补充 7 mL 完全培养基。</li>
		<li>以纵横交错的方式摇动培养皿以确保均匀混合，并在 37 °C 和 5% CO2 的 CO2 培养箱中培养。 注:完全培养基由补充有 10% 胎牛血清、1% 青霉素和链霉素的 DMEM 培养基组成。</li>
	</ol></li>
	<li>细胞活力评估<ol><li>选择对数生长期具有良好细胞状态的 MCF-7 细胞进行实验。在对数生长期，细胞通常表现出较短的分裂周期和较高的细胞分裂频率。<ol><li>使用电子显微镜观察 MCF 细胞的形态和生长速率。当细胞呈现均匀而密集的单层形态，并且细胞增殖明显时，可以确定细胞基本上处于对数生长期。</li>
		</ol></li>
		<li>细胞洗涤:用移液管从培养皿中取出培养基，加入 1 mL PBS 缓冲液洗涤细胞，然后弃去 PBS。</li>
		<li>细胞分离:加入 1 mL 胰蛋白酶洗涤细胞，等待约 1 分钟，让胰蛋白酶将细胞从培养皿中完全分离。轻轻敲击培养皿;观察细胞运动 in sheets 表示细胞彻底分离。</li>
		<li>终止细胞分离和细胞计数:加入 8 mL 完全培养基以终止分离，轻轻移液细胞，并使用细胞计数仪测定细胞悬液浓度。 注:在计数前，轻轻上下吹打，确保细胞悬液均匀。</li>
		<li>细胞接种:调整稀释的细胞悬液，使细胞密度达到每 100 μL 5,000 个细胞。使用移液枪将细胞悬液接种到 96 孔板的 21 个孔中，向每个孔中分配 100 μL。将 100 μL PBS 缓冲液添加到接种孔的周围孔中，以防止培养基过度蒸发。</li>
		<li>含 H2O2 的培养基的制备:在 12 孔细胞培养板上选择 9 个孔，并根据 H2O2 浓度的梯度（0、0.25、0.50、0.75、1.0、1.5、2.0 mM）分配标记。向标记为 2.0 mM 和 1.5 mM 的孔中加入 720 μL 完全培养基，并向其余孔中加入 360 μL 完全培养基。<ol><li>使用移液管，将 1.63 μL 和 1.22 μL 的 3% H2O2 试剂分别分配到标记为 2.0 mM 和 1.5 mM 的孔中。充分混合溶液。使用移液管将 360 μL 的 2.0 mM H2O2 转移到标记良好的 1.0 mM 中并混合;然后，将 360 μL 的 1.0 mM H2O2 转移到标记良好的 0.5 mM 中并混合;最后将 360 μL 的 0.5 mM H2O2 转移到标记良好的 0.25 mM 中。将 360 μL 的 1.5 mM H2O2 移液到标记良好的 0.75 mM 中以稀释并达到所需的浓度。（图 1）。 注意:确保移液器中的 H2O2 储备液完全转移到孔中，以避免任何残留溶液。在进行稀释之前，确保溶液充分混合。</li>
		</ol></li>
		<li>孵育:细胞接种约 24 小时后，一旦细胞粘附到表面，使用移液器从 96 孔板中取出培养基。根据浓度梯度，向每个孔中加入相应的含 H2O2 的培养基。将板放回培养箱中 12 小时。</li>
		<li>添加 CCK-8 试剂:在指定的 12 小时处理期后，使用移液器从 96 孔板中取出培养基，向每个孔中加入 100 μL 完全培养基和 10 μL CCK-8 试剂，并包括三个空白对照孔。将板在 37 °C 下孵育 2 小时。 注意:在添加样品的过程中，请避免形成气泡，以防止对 OD 值读数产生任何干扰。</li>
		<li>吸光度测量:从培养箱中取出 96 孔板，使用酶标仪测量 450 nm 波长的吸光度，并记录结果。</li>
		<li>根据IC50 的计算公式， 抑制率（%）=（药物实验组OD-药物对照孔平均OD）/（细胞对照孔平均OD-药物对照孔平均OD）×100% 将结果计算为对照培养物中吸光度的百分比。</li>
		<li>将处理后的实验数据导入统计分析软件，选择分析类型 非线性回归，构建四参数 logistic 模型，然后单击 拟合曲线 按钮进行曲线拟合。拟合过程之后，软件将自动计算 IC50 值。</li>
	</ol></li>
	<li>H2O2 诱导的 MCF-7 细胞氧化实验<ol><li>检索对数生长期具有良好细胞状态的 MCF-7 细胞以进行实验。</li>
		<li>洗涤，进行细胞分离，终止分离，并对细胞进行计数（有关具体方法，请参阅步骤 1.2）。</li>
		<li>细胞接种:将细胞悬液的密度调整为每培养皿 1 x 106 个细胞，每 6 cm 直径培养皿 4 mL 悬浮液。</li>
		<li>将两个 5 mL 微量离心管标记为 0.25 mM 和 0.75 mM 3% H2O2。向每个试管中加入 4 mL 完全培养基，然后分别加入 1.13 μL 和 3.40 μL 的 3% H2O2。轻轻摇动试管以确保充分混合。用含有 0、0.25 和 0.75 mM 的 H2O2 浓度的完全培养基替换三个培养皿中的培养基。</li>
		<li>孵育培养物:将培养皿放回培养箱中 12 小时。</li>
	</ol></li>
</ol>2. 细胞样品的制备和 ELISA 试剂的制备
<ol><li>细胞提取物样品的收集<ol><li>样品采集:弃去 MCF-7 细胞培养上清液，冲洗，进行细胞分离，终止分离，并收集细胞。将细胞悬液转移至 15 mL 离心管中。</li>
		<li>离心:以 800 x g 离心 5 分钟以收集细胞沉淀。添加 200 μL PBS 以重悬每个细胞样品，并通过重复冻融循环破坏细胞。将细胞裂解物以 1,500 x g 离心 10 分钟，并使用移液管收集上清液进行分析。 注:当细胞量低时，减少用于重悬的 PBS 体积。如果无法立即分析，样品可在 4 °C 下储存长达 1 周。对于长期储存，根据一次性使用量将样品分装并储存在 -80 °C 以避免重复冻融循环。</li>
	</ol></li>
	<li>ELISA 试剂的制备<ol><li>试剂解冻:让 ELISA 试剂（包括密封膜、预包被板、标准品、样品稀释剂、检测抗体-HRP、显色底物、终止液、浓缩洗涤缓冲液 （20x） 和测试样品达到室温 （18-25 °C） 20 分钟，然后再开始检测。使用前 15 分钟预打开 ELISA 读数仪。</li>
		<li>洗涤缓冲液的制备:计算所需体积的稀释洗涤缓冲液，然后用蒸馏水或去离子水将 20 倍浓缩洗涤缓冲液稀释成 1 倍工作溶液。将任何未使用的浓缩洗涤缓冲液放回 4 °C 冰箱中。 注意:洗涤缓冲液在使用前应新鲜制备。如果储存在冰箱中的浓缩洗涤缓冲液中存在晶体，请使其达到室温并轻轻摇晃，直到晶体完全溶解后再使用。</li>
		<li>标准品的编制<ol><li>制备浓度分别为 1.25、2.5、5、10、20 和 40 ng/mL 的标准样品的工作溶液。为保证实验结果的准确性，使用前应小心地上下混合标准样品，以避免在溶出过程中形成气泡。</li>
		</ol></li>
	</ol></li>
</ol>3. ELISA 实验
<ol><li>板涂层:提前设计标准品、样品和空白/对照品的孔总数，并取出所需的板条。向反应孔中加入 50 μL 标准工作溶液和不同浓度的检测样品。一式三份执行标准品，避免将任何样品添加到空白/对照孔25 中。 注意:ELISA 测定的实验步骤如图 2 所示。如果样品浓度超过检测范围，样品应在取样前用样品稀释剂稀释。添加样品时，将其添加到板底部，避免接触孔壁，轻轻摇晃混合，避免形成气泡。为确保实验的准确性，每次样品添加应在 10 分钟内完成。</li>
	<li>孵育:除空白孔外，向每个反应孔中加入 100 μL 辣根过氧化物酶 （HRP） 偶联的检测抗体，用板密封密封孔，并在 37 °C 下在恒温培养箱中孵育 60 分钟。</li>
	<li>洗涤:弃去液体，抖掉板孔中的液体，在吸水纸上拍干，向每个反应孔中加入 350 μL 洗涤缓冲液，静置 1-2 分钟，然后丢弃洗涤缓冲液。重复洗涤过程 5 次。 注意:彻底清洗至关重要，因为它直接影响实验结果的准确性。确保每次洗涤后洗涤缓冲液完全抖落。洗涤后小心擦去板底部的残留物，以免影响吸光度测量。</li>
	<li>显色反应:向每个反应孔中加入 50 μL 底物 A 和 50 μL 底物 B，密封板并在 37 °C 下避光孵育 15 分钟。 注:添加显色剂后，及时观察反应孔中的颜色变化。如果颜色太深，请添加终止液以提前终止反应。</li>
	<li>终止反应并测量吸光度:向每个反应孔中加入 50 μL 终止液，并立即用 ELISA 读数仪测量 450 nm 波长处的 OD 值（15 分钟内）。</li>
	<li>数据分析:将处理后的实验数据导入统计分析软件中，选择分析类型 Linear Regression。通过将 X 轴上标准品的浓度与 Y 轴上的光密度 （OD） 值作图来构建校准曲线。利用建立的校准曲线来确定样品中存在的 8-oxo-dG 的浓度。构建一个 Logistic 数学模型，然后单击 Fit Curve 按钮进行曲线拟合。在拟合过程之后，软件会自动计算标准曲线和 P 值。 注意:标准曲线不能重复使用，需要为每个实验重新确定。</li>
</ol>

确定 MCF-7 细胞中 DNA 氧化损伤的程度

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S., Islam, R., Ahmed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+gold+nanoparticles+in+ELISA,+PCR,+and+immuno-pcr+assays:+A+review.">Applications of gold nanoparticles in ELISA, PCR, and immuno-pcr assays: A review.</a> Anal Chim Acta. 1143, 250-266 (2021).</li><li>Berlina, A. N., Zherdev, A. V., Dzantiev, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA+and+lateral+flow+immunoassay+for+the+detection+of+food+colorants:+State+of+the+art.">ELISA and lateral flow immunoassay for the detection of food colorants: State of the art.</a> Crit Rev Anal Chem. 49 (3), 209-223 (2019).</li><li>Amber, K. T., Bloom, R., Hertl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+with+pooled+analysis+of+clinical+presentation+and+immunodiagnostic+testing+in+mucous+membrane+pemphigoid:+Association+of+anti-laminin-332+IGG+with+oropharyngeal+involvement+and+the+usefulness+of+ELISA.">A systematic review with pooled analysis of clinical presentation and immunodiagnostic testing in mucous membrane pemphigoid: Association of anti-laminin-332 IGG with oropharyngeal involvement and the usefulness of ELISA.</a> J Eur Acad Dermatol Venereol. 30 (1), 72-77 (2016).</li><li>Gervay, J., Mcreynolds, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+elisa+technology+to+measure+biological+activities+of+carbohydrates+relevant+in+disease+status.">Utilization of elisa technology to measure biological activities of carbohydrates relevant in disease status.</a> Curr Med Chem. 6 (2), 129-153 (1999).</li><li>Tabatabaei, M. S., Ahmed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assay+(ELISA).">Enzyme-linked immunosorbent assay (ELISA).</a> Methods Mol Biol. 2508, 115-134 (2022).</li><li>Aydin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+history,+principles,+and+types+of+ELISA,+and+our+laboratory+experience+with+peptide/protein+analyses+using+ELISA.">A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA.</a> Peptides. 72, 4-15 (2015).</li><li>Hayrapetyan, H., Tran, T., Tellez-Corrales, E., Madiraju, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+immunosorbent+assay:+Types+and+applications.">Enzyme-linked immunosorbent assay: Types and applications.</a> Methods Mol Biol. 2612, 1-17 (2023).</li><li>Eldahshoury, M. K., Hurley, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+sandwich+ELISA+to+detect+the+adulteration+of+human+breast+milk+by+cow+milk.">Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk.</a> J Dairy Sci. 106 (9), 5908-5915 (2023).</li><li>Tsumuraya, T., Hirama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationally+designed+synthetic+haptens+to+generate+anti-ciguatoxin+monoclonal+antibodies,+and+development+of+a+practical+sandwich+ELISA+to+detect+ciguatoxins.">Rationally designed synthetic haptens to generate anti-ciguatoxin monoclonal antibodies, and development of a practical sandwich ELISA to detect ciguatoxins.</a> Toxins (Basel). 11 (9), 533 (2019).</li><li>Ito, E., Iha, K., Yoshimura, T., Nakaishi, K., Watabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+diagnosis+with+ultrasensitive+elisa).">Early diagnosis with ultrasensitive elisa).</a> Adv Clin Chem. 101, 121-133 (2021).</li><li>Larsson, P., Parris, T. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+cell+viability+assays+for+drug+sensitivity+screens.">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/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>

作者没有需要披露的利益冲突。

ELISA 方法的开发对于现有和新的 DNA 损伤检测方法都非常重要。与传统的 HPLC 和质谱技术相比，这种方法不仅用户友好，而且灵敏度高，满足高通量筛选的要求30。这使得在大规模疾病筛查研究中监测 8-oxo-dG，有助于更深入地了解该生物标志物与各种疾病之间的相关性。
在实验过程中，几个关键步骤对于确保结果的可靠性和可重复性至关重要。最初，实验熟?...

DNA 氧化损伤是活性氧 （ROS） 的产生与细胞抗氧化防御系统之间不平衡的结果1。它主要涉及 DNA 嘌呤和嘧啶碱基的氧化 2,3。DNA 碱基的这种氧化修饰不仅损害了基因组的完整性，而且还包含广泛的病理问题，包括癌症、神经退行性疾病和心血管疾病 4,5。DNA 中的鸟嘌呤碱基具有最低的还原电位，并且最易氧化6。因此，8-氧代-7,8-二氢-2'-脱氧鸟苷 （8-oxo-dG） 是评估 DNA 氧化损伤程度的主要标志物 7,8。8-oxo-dG 的准确定量已成为解决疾病发生、进展和评估多因素氧化负荷的各个方面的关键问题9。
检测 8-oxo-dG 的传统方法，如高效液相色谱-电化学检测 （HPLC-ECD）、质谱和相关联用技术，表现出高灵敏度和特异性 10,11,12。然而，这些技术通常具有复杂的操作要求和高成本，这阻碍了它们在高通量样品分析中的广泛适用性和实用性。随着科学技术的不断进步，出现了各种新颖、高效、准确的方法。这些新技术的应用使我们能够更准确地量化 8-oxo-dG 的水平，并为深入研究氧化应激与疾病之间的关联提供更有力的工具。例如，研究人员应用纳米孔技术对 DNA 进行定量检测和测序13，使用单击式代码测序策略识别 DNA 损伤类型14，开发高通量测序方法，并通过将生物素-链霉亲和素与 ELISA 整合来创建基于 8-oxoG 的生物传感器15。其中，ELISA 在特异性、高通量筛选和成本方面具有公认的优势，是 8-oxo-dG 检测的理想解决方案之一。因此，开发一种高通量、高灵敏度、方便、快速的 8-oxo-dG 检测方法至关重要。
酶联免疫吸附测定 （ELISA） 技术开发于 1971年 16 年，在过去 50 年中迅速发展，现已成为生物学和医学领域最常用的检测方法之一 17,18,19。ELISA 技术具有高灵敏度和特异性，反应时间短，易于使用，是大规模样品检测和高通量分析的广泛认可选择20。因此，ELISA 已广泛用于细胞内化合物、蛋白质、抗体或分子的定量或半定量分析 21,22,23。例如，它已被用于检测与各种疾病、药物残留和生物分子相关的生物标志物24。根据实验设计和原理，ELISA 可分为四种主要类型25。这些方法包括直接 ELISA、间接 ELISA、夹心 ELISA 和竞争性 ELISA26,27。其中，夹心 ELISA 利用两种特异性抗体，一种用于捕获靶分子，另一种用于检测，被选中用于本文的研究。夹心 ELISA 的实验原理如下:首先，将特异性抗体固定在微孔板的孔中以捕获目标分析物。添加标准品或样品后，目标分析物与固定化抗体结合。随后，加入识别抗原上不同表位的标记抗体，形成夹心结构。去除未结合的抗体后，添加底物。在二抗的催化作用下，发生有色反应，颜色的强度与样品中目标分析物的浓度呈正相关。最后，测量光密度 （OD） 以确定样品的浓度。夹心 ELISA 具有对目标样品的灵敏度和特异性更高等优点，这使其适用于检测低浓度的目标分析物和复杂样品28。此外，获得的结果可以量化以供进一步分析。这些因素使夹心 ELISA 成为科学研究和临床实验室中常用的检测方法29。
本研究旨在定量检测 MCF-7 细胞中的 8-oxo-dG，以确定细胞中 DNA 氧化损伤的程度。本研究包括两个主要部分:构建 MCF-7 细胞 DNA 氧化损伤模型和使用 ELISA 检测 8-oxo-dG。首先，在体外培养 MCF-7 细胞，并用不同浓度的 H2O2 处理不同的持续时间。使用 CCK-8 测定法评估细胞活力，以确定 MCF-7 细胞中 H2O2 的半数最大抑制浓度 （IC50）。根据 IC50 值，选择合适的 H2O2 处理时间和诱导浓度。为了提取因氧化损伤而受损的 MCF-7 细胞样品，获得细胞样品和上清液，并将其添加到先前涂有 8-oxo-dG 抗体的酶联孔中。样品中存在的 8-oxo-dG 将与与固相载体结合的抗体结合。然后，加入用辣根过氧化物酶标记的 8-oxo-dG 抗体。将反应混合物在恒温下孵育，以确保样品与抗体完全结合。洗涤除去未结合的酶，然后加入比色底物，产生蓝色。在酸的作用下，溶液变黄。最后，在 450 nm 处测量反应孔样品的 OD 值，样品中 8-oxo-dG 的浓度与 OD 值成正比。通过生成标准曲线，可以计算样品中 8-oxo-dG 的浓度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>
		</tr>
	</tbody>
</table>

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

8-氧代-7,8-二氢-2'-脱氧鸟苷 （8-oxo-dG） 碱基是常见的 DNA 氧化损伤的主要形式。DNA 损伤对基因表达产生深远影响，是刺激神经退行性疾病、癌症和衰老的关键因素。因此，8-oxoG 的精确定量在 DNA 损伤检测方法的研究中具有临床意义。然而，目前，现有的 8-oxoG 检测方法在便利性、便利性、可负担性和更高的灵敏度方面提出了挑战。我们采用夹心酶联免疫吸附测定 （ELISA） 技术，一种高效、快速的比色法，检测用不同浓度的过氧化氢 （H2O2） 刺激的 MCF-7 细胞样品中 8-oxo-dG 含量的变化。我们通过检测 MCF-7 细胞中诱导 MCF-7 细胞氧化损伤的 H2O2 浓度50 来确定其浓度。随后，我们用 0、0.25 和 0.75 mM H2O2 处理 MCF-7 细胞 12 小时，并从细胞中提取 8-oxo-dG。最后，对样品进行 ELISA。经过一系列步骤，包括铺板、洗涤、孵育、显色、终止反应和数据收集，我们成功检测到 H2O2 诱导的 MCF-7 细胞中 8-oxo-dG 含量的变化。通过这些努力，我们的目标是建立一种评估细胞样本中 DNA 氧化损伤程度的方法，并在此过程中推动开发更方便、更方便的 DNA 损伤检测方法。这项工作有望为探索 DNA 氧化损伤与各个领域之间的关联分析做出有意义的贡献，包括疾病的临床研究和有毒物质的检测。

如图 3 所示，X 轴表示应用于 MCF-7 细胞的 H2O2 浓度，而 Y 轴表示细胞活力。用 0.75 mM 处理 12 小时将 MCF-7 细胞的活力降低至 67%。伴随着浓度的增加，MCF-7 细胞的活力显着降低，尤其是在 1.5 mM 的浓度下，活力下降到 3% 以下（表 1）。实验结果表明 MCF-7 细胞的 IC50 为 0.7655 mM。
在这一系列实验中，我们采用了增强的 ELIS...

作者聚焦:使用 ELISA 定量评估 MCF-7 细胞中 8-oxo-dG

该方案描述了一种通过 ELISA 技术定量检测 MCF-7 细胞中 DNA 氧化损伤的有效方法。

使用 ELISA 测定定量 8-oxo-dG 水平以评估 MCF-7 细胞中的氧化 DNA 损伤

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 ...

本研究旨在使用 ELISA 定量测量 MCF-7 细胞中的 8-oxo-dG，评估过氧化氢对 DNA 氧化损伤的影响。具体来说，本视频中详细介绍了 ELISA 步骤以提高效率。检测 8-oxo-dG 的技术，包括高效液相色谱法和电化学检测、质谱法和其他复杂的方法，已证明其疗效。但是，这些方法通常涉及许多繁琐的步骤，并且可能非常昂贵。该方案解决了在高效和灵敏地检测氧化 DNA 损伤方面的研究空白，特别是细胞样品中的 8-oxo-dG，这对于了解其对疾病、衰老和对环境压力的反应的影响至关重要。我的实验室专注于 DNA 损伤事件检测的方法创新。这将使我们能够对 DNA 损伤标记进行深入的实验室科学研究。

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

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JoVE Journal

Biochemistry

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

732 次观看.  Suzhou Vocational Health College. 本研究旨在使用 ELISA 定量测量 MCF-7 细胞中的 8-oxo-dG，评估过氧化氢对 DNA 氧化损伤的影响。具体来说，本视频中详细介绍了 ELISA 步骤以提高效率。检测 8-oxo-dG 的技术，包括高效液相色谱法和电化学检测、质谱法和其他复杂的方法，已证明其疗效。但是，这些方法通常涉及许多繁琐的步骤，并且可能非常昂贵。该方案解决了在高效和灵敏地检测氧化 DNA 损伤方面的研究?...

视频: 作者聚焦:使用 ELISA 定量评估 MCF-7 细胞中 8-oxo-dG

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.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

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

科研

生物化学

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.

滴定 ELISA 法测定受体配体相互作用的离解常数

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

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.

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

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.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

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.

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

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

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

测量天然产品生物调节特性的平面运动分析

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦:基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...