Name: experimental protocol for detecting mitochondrial function in hepatocytes exposed to organochlorine pesticides
Uploaded: 2020-09-16T12:30:00
Description: Watch this Scientific Journal Video about Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides at JoVE.com

这项工作得到了国家自然科学基金（第81573174号，81570574号）的支持;江苏省杰出青年基金（SBK2014010296）;中国教育部研究项目（213015A）;江苏高等教育机构优先学术项目发展，江苏高等教育院校旗舰专业发展;环境化学和生态毒理国家重点实验室开放项目项目（KF2015-01）。

所有实验和实验规程都按照有关准则和规定进行，并经南京医科大学地方伦理委员会批准。
1. TEM 的线粒体超结构
<ol><li>收集 HepG2 细胞<ol>
<li>种子 HepG2 细胞在 100 毫米的菜肴。储存在37°C和5%CO22。</li>
		<li>1.5 mLEP 管中具有 0.25% EDTA 的消化细胞。</li>
		<li>在室温 （RT） 下以 1000 x g 的离心机 3 分钟。丢弃上一杯。</li>
		<li>收集 4-6 x 105 HepG2 细胞。</li>
	</ol>...

<ol>
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J., 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=Persistent+organochlorinated+pesticides+and+mechanisms+of+their+toxicity.">Persistent organochlorinated pesticides and mechanisms of their toxicity.</a> Toxicology. 307, 74-88 (2013).</li><li>Dirinck, E., 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=Obesity+and+persistent+organic+pollutants:+possible+obesogenic+effect+of+organochlorine+pesticides+and+polychlorinated+biphenyls.">Obesity and persistent organic pollutants: possible obesogenic effect of organochlorine pesticides and polychlorinated biphenyls.</a> Obesity (Silver Spring). 19 (4), 709-714 (2011).</li><li>Genini, D., 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=Mitochondrial+dysfunction+induced+by+a+SH2+domain-targeting+STAT3+inhibitor+leads+to+metabolic+synthetic+lethality+in+cancer+cells.">Mitochondrial dysfunction induced by a SH2 domain-targeting STAT3 inhibitor leads to metabolic synthetic lethality in cancer cells.</a> Proc Natl Acad Sci U S A. , (2017).</li><li>Korovljev, D., Trivic, T., Drid, P., Ostojic, S. 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检测协议成功的关键是使用各种实验方法，这些实验方法涵盖了从表型到机制的研究。在这项研究中，HepG2细胞在DMEM中培养青霉素和链霉素和10%胎儿牛血清。当细胞达到40-50%汇合时，添加+-HCH（0，10，100纳克/mL）并孵育24小时。我们首先使用TEM，它显示了代表性的OCPs引起的肝细胞的超结构变化，+-HCH，显示了线粒体结构的损伤（图1）。)此外，还进行...

有机氯农药（OCPs）对健康的影响，如生殖干扰、免疫毒性、代谢变化等，此前已研究过1、2、3。,2,3检测细胞代谢和发现线粒体功能障碍的方法使科学家能够理解线粒体功能的作用（即：，线粒体STAT3水平，乳酸，丙酮酸盐，乳酸与丙酮酸盐比，共酶Q10，线粒体质子泄漏，生物能量，生物发生和动力学）在老化，肥胖，糖尿病，心血管功能，癌症和安全毒性4,54，5，6，7,6等领域7。本文介绍了评估OCPs引起的线粒体功能障碍的方法。
我们向+-六氯环氧烷（+-H环环）暴露，一种具有代表性的OCPs，剂量相当于人体内部接触24小时。首先，TEM用于观察肝细胞的超结构，如核、线粒体和内质电8。与普通显微镜相比，TEM能够探索细胞和细胞成分（细胞系或组织）的二、三D超结构、形态、化学成分以及在现代科学技术中起关键作用的自然或人工材料的功能。线粒体功能通过线粒体荧光强度、腺苷5'-三磷酸（ATP）水平、耗氧率（OCR）和线粒体膜电位（MMP）进一步评估，在使用β-HCH孵育的HpG2细胞中。Mito-跟踪器绿色是一种线粒体绿色荧光探针，可用于活细胞线粒体特异性荧光染色。肝细胞中的线粒体被线粒体染色的线托跟踪绿色溶液和线粒体荧光强度，数量和模式观察到与共理显微镜9。线粒体绿色荧光探针可用于染色活细胞。与罗丹胺123或JC-1相比，线粒体绿色荧光探针不依赖于线粒体染色的线粒体膜潜力。ATP水平由荧光素-荧光素试剂盒确定，并由蛋白质浓度进行规范化。ATP 检测试剂盒可用于检测常见溶液、细胞或组织中的 ATP 水平。此试剂盒基于萤火虫荧光素催化的荧光酶，当需要 ATP 提供能量时，可产生荧光。当萤火虫荧光酶和荧光素过量时，在一定浓度范围内，荧光的产生与ATP的浓度成正比。此外，该套件还经过专门设计，以优化 ATP 的化学发光。ATP作为最重要的能量分子，在细胞的各种生理或病理过程中起着重要的作用。ATP水平的变化可以反映细胞功能的缺陷，特别是线粒体能量的产生。通常在凋亡、坏死或处于一定毒性状态下，细胞ATP水平降低 10。JC-1 的 MSP 检测套件是使用 JC-1 作为荧光探针的试剂盒，可快速、灵敏地检测细胞、组织或纯化 MMP。它可用于早期发现凋亡。阳离子染料JC-1是一种荧光探针，用于检测MMP，可根据绿色和红色荧光比的变化在流式细胞仪下进行分析。当 MMP 高时，JC-1 在线粒体的基质中聚合，形成聚合物（J-聚合），产生红色荧光。当MMP低时，JC-1不能积累，形成单体产生绿色荧光11。因此，红与绿荧光的比例可以反映MMP的水平。细胞的OCR是正常细胞功能12的关键指标。它是研究线粒体功能的参数。细胞线粒体压力测试试剂盒为分析线粒体功能的关键参数提供了一种稳定的方法。该试剂盒提供质量控制和预测试剂，以及执行细胞线粒体应激测试的标准方法。它可用于检测所有细胞类型，包括原细胞、细胞系、悬浮细胞，也可用于小岛、线虫、酵母和孤立的线粒体。
OCR 测量可以提供对细胞的生理状态或变化的宝贵见解。它由细胞外通量分析仪确定，以检测呼吸基线、质子泄漏、最大呼吸、ATP 周转量和储备能力。简言之，在对OCR进行基线测量后，每井依次添加寡霉素（ATP，FCCP（线粒体氧化磷酸化未结合）和抗霉素甲/罗酮（氧气消耗抑制剂）后，检测到OCR。
为了促进在体外检测肝细胞中线粒体功能的更具体的方案，我们在此介绍TEM、共生显微镜、发光计、流细胞仪和细胞外通量分析仪的实验，并在未来用于线粒体损伤相关不良结果研究。

<table border="0" cellpadding="0" cellspacing="0" style="width:756px;" width="755">
	<tbody>
		<tr height="21">
			<td>Transmission electron microscope&#160;</td>
			<td>FEI</td>
			<td>Tecnai G2 Spirit Bio TWIN</td>
			<td>High-contrast, high-resolution imaging, Low-dose observation and imaging, Low-temperature observation, Outstanding analytical performance, Automation for convenience and performance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">Mito-Tracker Green</td>
			<td style="width:69px;">Beyotime</td>
			<td style="width:161px;">C1048</td>
			<td style="width:320px;">Mito-Tracker Green is a mitochondrial green fluorescent probe that can be used for live cell mitochondrial-specific fluorescent staining.</td>
		</tr>
		<tr height="21">
			<td height="105" style="height:105px;width:205px;">Laser scanning confocal microscope</td>
			<td style="width:69px;">Zeiss</td>
			<td style="width:161px;">700B</td>
			<td style="width:320px;">The design is compact, stable, light path is the shortest, high light precision, creative technology and sophisticated scanning technology together to&#160; produce a perfect 3-dimensional specimen image.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:205px;">Enhanced ATP Assay Kit</td>
			<td style="width:69px;">Beyotime</td>
			<td style="width:161px;">S0027</td>
			<td style="width:320px;">Enhanced ATP Assay Kit can be used to detect ATP (adenosine 5&#39;-triphosphate) levels in common solutions, cells or tissues. Cells and tissue samples can be split to complete the sample preparation, detection sensitivity up to 0.1nmol / L, chemiluminescence can be sustained for 30 minutes.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:205px;">Luminometer</td>
			<td style="width:69px;">Berthold</td>
			<td style="width:161px;">Centro LB 960</td>
			<td style="width:320px;">Luminometer is chemiluminescence detector, the test sample itself can be light, do not need to stimulate. Luminometer is the instrument that detects chemiluminescence.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">BCA Protein Assay Kit</td>
			<td style="width:69px;">Beyotime</td>
			<td style="width:161px;">P0012</td>
			<td style="width:320px;">BCA Protein Assay Kit is one of the most commonly used methods for detecting protein concentrations.</td>
		</tr>
		<tr height="21">
			<td height="42" style="height:42px;width:205px;">Multimode reader</td>
			<td style="width:69px;">TECAN</td>
			<td style="width:161px;">InfiniteM200</td>
			<td style="width:320px;">Multimode reader be used to detect protein consentration.</td>
		</tr>
	</tbody>
</table>

experimental protocol for detecting mitochondrial function in hepatocytes exposed to organochlorine pesticides

本文提出了检测肝线粒体功能的详细方法，以便更好地了解肝细胞中环境有机氯农药（OCPs）引起的代谢紊乱的原因。HepG2细胞在同等剂量的内部接触下，在24小时内暴露在β-六氯环丙烷（+-六氯环丙烷）中。通过透射电子显微镜（TEM）对肝细胞的超结构进行了检查，以显示线粒体的损伤。线粒体功能通过线粒体荧光强度、腺苷5'-三磷酸（ATP）水平、氧消耗率（OCR）和线粒体膜电位（MMP）进一步评估，这些细胞在使用β-HCH孵育的HpG2细胞中。线粒体荧光强度后染色线粒体绿色荧光探针观察到荧光显微镜。荧光素-荧光酶反应用于确定ATP水平。在流式细胞学下检测出MMP。使用细胞外通量分析仪测量OCR。总之，这些协议用于检测肝细胞中的线粒体功能，以调查线粒体损伤。

HepG2细胞暴露在+-六氯上细胞中的线粒体受到明显损伤。散落的线粒体有轻微扩张，形状不规则，线粒体脊消失，线粒体结构相对异常（图1）。
平均线粒体绿色荧光强度，代表线粒体，在接触+-HCH的 HepG2 细胞（图2）和ATP水平（图3） 中下降。荧光强度和ATP水平...

Watch this Scientific Journal Video about Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides at JoVE.com

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

了解环境有机氯农药（OCPs）对肝细胞线粒体功能的影响，对于探讨OCP引起代谢紊乱的机理十分重要。本文提出了检测肝线粒体功能的详细方法。

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

这是一个混乱的调用回答一个关键问题，你也有一个领域的功能，如ATP水平和氧气消耗率。新的或一页旧，这种技术是，它很容易执行，它是一个细胞线粒体功能的特殊评估很多。为了开始这个实验，在玻璃底部培养皿中播种2万个 HepG2 细胞。在37摄氏度的培养箱中加入β-六氯环氧烷或B-HCH，24小时加入5%的二氧化碳。通过将无水DMM细胞培养基中添加无水DMSO，准备一毫摩尔线粒体绿色荧光探针溶液。然后将溶液每盘一毫升添加到玻璃底部培养皿中的细胞中。并在37摄氏度下孵育45分钟。孵育后，从细胞中去除线粒体绿色荧光探针溶液。在37摄氏度的DMEM细胞培养基下，每盘新鲜准备的菜中加入一毫升。要检测线粒体中的绿色荧光，在共和显微镜下观察细胞。为了评估细胞ATP的水平，种子在6个井板中的20，000个 HepG2细胞。加入乙基六氯多氯二羊，在37摄氏度下孵育24小时。向每个井的细胞中加入200微升的解液缓冲液。在摄氏4度下以12，000次G离心5分钟，然后按照文本协议中所述进行收集上一代。向板中加入 100 微升 ATP 检测缓冲液。在室温下将100微升收集的细胞上一提液添加到96井板中，然后按照文本协议中描述的发光计检测细胞ATP。为了评估线粒体膜电位，如前所述，在六个井板中生长 HepG2 细胞，并添加乙基六氯二线。在37摄氏度下用0.5毫升0.25%EDTA消化细胞，收集细胞，一分钟。加入一毫升DMEM停止消化，并将消化的细胞转移到1.5毫升管。将管子在1000倍G下离心三分钟，然后丢弃上流液。用0.5毫升细胞培养基和0.5毫升JC1线粒体膜电位染料稀释细胞颗粒。在37摄氏度下孵育20分钟。接下来，在4摄氏度下以600倍G离心管3分钟，然后根据文本协议清洗细胞颗粒。离心冲走洗颗粒后，取出上经剂。并在0.5毫升JC1染色缓冲液中重新悬浮颗粒。通过流式细胞分析 JC1 染色细胞，检测绿色和红色荧光。检测到 JC1 单体时，将激励光设置为 490 纳米，将发射光设置为 530 纳米。检测到 JC1 聚合物时，将激发光设置为 525 纳米，将发射光设置为 590 纳米。为了分析耗氧率，将 HepG2 细胞在 96 井细胞培养板中播种，密度为每 100 微升 4，500 个细胞。在37摄氏度下孵育24小时后，确保每一个井中的细胞都覆盖着中等的细胞。在运行 OCR 软件之前，请将带墨盒的微孔板和细胞培养板加载到文本协议中描述的细胞外通量分析仪中。打开 OCR 软件并在应用下拉菜单中，选择细胞 mito 压力测试套件，然后单击"启动应用"按钮。接下来，单击运行压力测试按钮。当出现细胞压力测试屏幕时，在细胞种子编号框中输入每井播种的细胞数量，以及基底细胞OCR框中的平均OCR。输入每个试剂的最终工作浓度，然后注射试剂。单击下一个按钮。当出现组信息屏幕时，通过选择颜色并指定名称，将组分配给未分配的井。然后单击相应的油井。单击下一个按钮，在显示的压力测试注入布局屏幕中，单击"开始"以在分析仪上运行压力测试。完成运行后，按照软件中的提示操作，然后取出并丢弃墨盒和电池板。线粒体绿色荧光染色的平均强度在接触β-HCH的HpG2细胞中降低。这表明线粒体数量减少，受损线粒体增加，农药浓度增加。此外，HepG2细胞中的ATP水平随着乙氯二氯六基暴露浓度的增加而降低，表明这些细胞中线粒体的功能下降。JC1 染色线粒体膜电位（MMP）后，流式细胞仪在完整的 HepG2 细胞中显示高红色绿色 JC1 荧光。这是因为存在红色荧光JC1聚合体，这是高MMP的标志。在使用β-六氯危险症治疗的细胞中，红绿色JC1荧光由于存在绿色荧光JC1单体而减少，这是低MMP的标志。最后，随着乙氯二制六氯六基六氯基暴露浓度的增加，氧气消耗率也有所下降，进一步表明这种农药损害了适当的线粒体功能。哦，好吧，尝试这个程序，重要的是记住，看到细胞适合测试它。其发展，为基础功能领域的研究探索相关生物体的相关医学作用铺平了道路。观看此视频后，您应该对如何检测数量或质量、容量、现在的成员或喷口表下的潜在功能有一个很好的了解。

Research

JoVE Journal

Environment

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

用于操纵植物诱导土壤异质性的实验协议

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

科研

环境科学

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

一个协议，用于开展降雨模拟研究土壤径流

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

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Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

表征和被动采样的应用在农药水监测

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

实验协议生产生物柴油与饱和酮的分离从商业余积<em&gt;等鞭金藻</em&gt;藻类生物量

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

用于蜂蜜蜜蜂筛选化学成分的视频跟踪协议

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

纳米材料在水介质中的分散: 对协议优化的研究

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...