Name: mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay
Uploaded: 2018-05-01T12:30:00
Description: Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

这项研究得到了来自山东省杰出科学家 (JQ201421) 的赠款和自然科学基金 (81371226) 赠款的资助。

1. 线粒体的分离
注: 所有解决方案和设备应预换热器 0-4 摄氏度, 并保持在冰上。
<ol><li>种子约 3 x 106 SH-SY5Y 细胞每10厘米细胞培养皿。高糖 Dulbecco 修饰鹰培养基中培养细胞, 10% 胎牛血清, 青霉素 (100 U/毫升)-链霉素 (100 µg/毫升)。在包含 5% CO2的孵化器中保持37摄氏度。 注意: 每个隔离检测都需要至少 1-2 x 107 SH-SY5Y 单元格。</li>
	<li>取出培养基, 在10厘?...

<ol>
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K., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+calcium+indicators+underestimate+increases+in+intracellular+calcium+concentrations+associated+with+excitotoxic+glutamate+stimulations.">High-affinity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations.</a> Neuroscience. 89 (1), 91-100 (1999).</li><li>Bambrick, L. 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P., 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+free+calcium+levels+(Rhod-2+fluorescence)+and+ultrastructural+alterations+in+neuronally+differentiated+PC12+cells+during+ceramide-dependent+cell+death.">Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.</a> J Comp Neurol. 426 (2), 297-315 (2000).</li><li>Allman, R., Hann, A. C., Phillips, A. P., Martin, K. L., Lloyd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Azotobacter+vinelandii+with+correlation+of+Coulter+cell+size,+flow+cytometric+parameters,+and+ultrastructure.">Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.</a> Cytometry. 11 (7), 822-831 (1990).</li><li>Wiechelman, K. J., Braun, R. D., Fitzpatrick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+bicinchoninic+acid+protein+assay:+identification+of+the+groups+responsible+for+color+formation.">Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation.</a> Anal Biochem. 175 (1), 231-237 (1988).</li><li>Petronilli, V., Nicolli, A., Costantini, P., Colonna, R., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+permeability+transition+pore,+a+voltage-dependent+mitochondrial+channel+inhibited+by+cyclosporin+A.">Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.</a> Biochim Biophys Acta. 1187 (2), 255-259 (1994).</li><li>Pastorino, J. G., 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=Functional+consequences+of+the+sustained+or+transient+activation+by+Bax+of+the+mitochondrial+permeability+transition+pore.">Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.</a> J Biol Chem. 274 (44), 31734-31739 (1999).</li><li>Gunter, T. E., Sheu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+possible+functions+of+mitochondrial+Ca(2+)+transport+mechanisms.">Characteristics and possible functions of mitochondrial Ca(2+) transport mechanisms.</a> Biochim Biophys Acta. 1787 (11), 1291-1308 (2009).</li></ol>

在这里, 我们描述了一个简单有效的线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验的协议。
对于线粒体的隔离, 要确保所有的材料和管子都在冰上, 特别是当细胞均匀化时。0.25% 胰蛋白酶-EDTA 也可用于分离皿中的细胞5分钟, 在37摄氏度。15-25 冲程后, 必须观察显微镜下的完整细胞膜, 避免线粒体膜的断裂。然而, 我们获得的分离的线粒体是粗线粒体和纯?...

线粒体是主要的细胞器官, 以产生近95% 的 ATP 在哺乳动物细胞中使用氧化磷酸化。据报道, 通过线粒体、adp 和无机磷酸盐的存在, micromolar 的 Ca2 +的螯合浓度可以用来 phosphorylate adp 合成 ATP1。当胞浆 Ca2 +的浓度超过阈值时, 线粒体可以快速吸收 Ca2 +并缓慢地将其流出。因此, 功能线粒体可以流入增加胞浆 Ca2 +。与参与氧化磷酸化无关, 线粒体 Ca2 +也参与胞浆钙信号和激活线粒体凋亡机制, 诱导开放的 mPTP 在内线粒体膜2。人们普遍认识到, 胞内 Ca 的异常海拔2 +可以通过打开 mPTP3来诱发线粒体大面积肿胀。因此, 本协议旨在通过量化线粒体 Ca2 +保留容量和 Ca2 +-触发线粒体肿胀来评估线粒体功能。
Ca2 +保留容量是衡量线粒体吸收胞浆钙的能力。线粒体可以缓冲胞浆游离钙和调节钙依赖的细胞过程中的钙吸收形式的非活性沉淀。受损的 Ca2 +保留容量发生在与能源限制有关的压力现象中, 甚至神经退行性疾病4,5。分离的线粒体或 digitonin-透细胞可用于识别 Ca2 +保留容量, 并且由具有额外谷氨酸和苹果酸的孤立线粒体积累 Ca2 +的提升能力不受线粒体隔离过程6。滴定量的 digitonin 应用于 permeabilize 不同细胞类型的血浆膜。ca 的 hexapotassium 盐2 +-结合绿色荧光染料, 一个 Ca2 +敏感细胞-impermeant 可见光兴奋指示器, 已被广泛使用的7。Ca2 +-结合绿色荧光染料是一种低亲和力指示器, 用于表明胞内游离 Ca2 +浓度在长时间 (5 分钟) 刺激8中继续上升。该方法比放射性过滤技术灵敏度低, 但大大简化。额外的 Ca2 +提升了钙绿色荧光, 线粒体的 Ca2 +吸收将荧光返回到基线。连续添加了 ca2 + , 直到线粒体未能摄取 extramitochondrial Ca2 + 9。荧光微板块读取器可用于连续报告钙绿色荧光。
在 ca2 +累积之后, 线粒体偏振光, 将 Ca2 +释放到介质中, 并开始膨胀。Ca2 +触发的线粒体肿胀用于指示 mPTP 开口。电子显微镜和光吸收率降低 540 nm 可用于测量 Ca2 +触发的线粒体肿胀10,11。线粒体体积可以直接确定的前角光散射12, 其中吸收减少反映线粒体基质的被动肿胀。
在这里, 我们说明了检查线粒体 Ca2 +保留容量和 Ca2 +触发线粒体肿胀从 SH-SY5Y 细胞分离的线粒体的方法。

<table>
	<tbody>
		<tr>
			<td>SH-SY5Y cell line</td>
			<td>ATCC (The Global Bioresource Center)</td>
			<td>ATCC Number: CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium green-5N</td>
			<td>Life Technologies</td>
			<td>c-3737</td>
			<td>Ca2+-binding green fluorescent dye</td>
		</tr>
		<tr>
			<td>complete protease inhibitors</td>
			<td>Roche Molecular Biochemicals</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>glass homogenizer</td>
			<td>Kimble Chase</td>
			<td>9885303002</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>RES5063G-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>malate</td>
			<td>Sigma-Aldrich</td>
			<td>46940-U</td>
			<td></td>
		</tr>
		<tr>
			<td>rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>00457</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>V900050</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>V900266</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan flash instruments</td>
			<td>Thermo Scientific</td>
			<td>IC100E</td>
			<td></td>
		</tr>
		<tr>
			<td>Bongkrekate</td>
			<td>Biovision</td>
			<td>1820-100</td>
			<td></td>
		</tr>
		<tr>
			<td>atractyloside</td>
			<td>Sigma-Aldrich</td>
			<td>C4992</td>
			<td></td>
		</tr>
		<tr>
			<td>high-glucose Dulbecco&#8217;s modified Eagle medium</td>
			<td>Hyclone</td>
			<td>SH30022</td>
			<td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SV30087</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Invitrogen</td>
			<td>11860-038</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Thermo Scientific</td>
			<td>NCI3225CH</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay

氧化磷酸化生产 ATP 是线粒体的主要功能。高真核生物中的线粒体也参与胞浆 Ca2 +缓冲, 线粒体中的 ATP 生产可以通过 intramitochondrial 自由 Ca2 +浓度进行介导。Ca2 +保留容量可以被视为线粒体在线粒体基质中保留钙的能力。累积胞内 Ca2 +导致内线粒体膜通透性, 称为线粒体通透性过渡孔 (mPTP) 的开口, 导致分子量小于 1.5 kDa 的分子泄漏。Ca2 +触发的线粒体肿胀用于指示 mPTP 开口。在这里, 我们描述了两种检测, 以检查 ca2 +保留容量和 ca2 +触发线粒体肿胀在孤立的线粒体。添加了一定数量的 Ca2 +后, 所有步骤都可以在一天内完成, 并由微板块读取器记录。因此, 这两种简单而有效的检测方法可以用来评估与 Ca2 +相关的线粒体功能。

Ca2 +保留容量的代表性结果: 结果以荧光值表示。随着 Ca2 + (200 nmols/毫克线粒体蛋白) 的额外脉冲, 荧光量在基线上增加了两个 mPTP 开口。5微米 Bongkrekate (BKA), 一抑制剂的 ca2 +诱导 mPTP 开放, 或1微米苍术苷二 (ATR), 一个激活剂的ca2 + 诱导 mPTP 开放, 增加了孤立的线粒体。添加的 ca2 +的总量, 直到在基线荧光以上的双增量指示的 ...

Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验

本协议的目的是描述一种检查 ca2 +保留容量和 ca2 +触发的 SH-SY5Y 细胞线粒体肿胀的方法逐步。

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

Research

JoVE Journal

Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

在单个细胞中的线粒体DNA复制的可视化EDU信号放大

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

科研

神经科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

活大鼠皮层神经元线粒体膜电位和活性氧的测定

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

用于线粒体疾病建模的人脑类器官的生成

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

使用遗传编码的Ca2 + 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca2 + 摄取进行成像

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

Live-imaging of Mitochondrial System in Cultured Astrocytes

培养的星形胶质细胞中线粒体系统的实时成像

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and ...