Name: analyses of mitochondrial calcium influx in isolated mitochondria and cultured cells
Uploaded: 2018-04-27T13:30:00
Description: Watch this Scientific Journal Video about Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells at JoVE.com

这项工作得到了美国心脏协会 (J.Q.K.) 的赠款资助。

本议定书所述的所有方法均已由埃默里大学机构动物护理和使用委员会批准。
注: 第一部分是用平板阅读器测量线粒体 Ca2 +在孤立的心肌线粒体中的流入的实验程序。
1. 试剂和溶液
<ol><li>使500毫升的 MS EGTA 缓冲线粒体隔离: 225 毫米甘露醇, 75 毫米蔗糖, 5 毫米 4-(2-羟乙基)-1-piperazineethanesulfonic 酸 (HEPES), 1 毫米乙二醇-双 (β氨基乙基醚)-n-, n, n ', n '-?...

<ol>
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在这里, 我们描述了两种测量线粒体 Ca2 +流入的方法。基于印版阅读器的钙 green-5N 方法监视 extramitochondrial ca2 +级别, 是一个 ca2 +摄取分析, 非常适合在孤立的线粒体中测量。虽然我们已经显示了来自孤立小鼠心肌线粒体的代表性结果, 但这种检测方法可以很容易地适应线粒体丰富的组织线粒体, 包括肝脏、骨骼肌肉和大脑。此外, 对于传统用于 Ca2 +测量 (如 fluorometers...

线粒体是胞内 Ca2 +存储和信号传递的关键站点。数十年的研究表明, 线粒体有能力导入和隔离 Ca2 + 1,2。但是, 线粒体不仅仅是 Ca2 +存储的被动站点。在线粒体隔间的 Ca2 +执行基本信号功能, 包括调节代谢输出和激活线粒体介导的细胞死亡通路, 此前已对3进行了回顾。对于代谢调节, Ca2 +增强了 tricarboxylic 酸循环和呼吸链复合体的三基质局部化脱氢酶的活性, 以增加线粒体能量生产 4, 5.与线粒体 ca2 +过载和失调线粒体 ca2 +处理, Ca2 +触发线粒体通透性过渡孔 (MPTP) 开放, 导致线粒体内膜通透,膜电位损失, 线粒体功能障碍, 肿胀, 破裂最终, 细胞死亡6,7,8,9。因此, 线粒体 Ca2 +信号直接影响细胞生命和死亡通路通过新陈代谢控制和 MPTP 死亡轴。
近年来, 对线粒体 ca2 +动力学研究的兴趣迅速扩大, 大部分原因是线粒体 ca2 + uniporter 复合体的分子成分的鉴定, 线粒体内作为 Ca2 +的主要模式的膜传送器导入到线粒体矩阵10,11,12。uniporter 的这些结构和调控亚基的鉴定提出了基因靶向线粒体 Ca2 +流入调节线粒体功能和功能障碍的可能性, 并促进了研究uniporter 复合体和线粒体 Ca 的贡献2 +流入疾病13,14,15。事实上, 线粒体 Ca2 +信号已牵连到多种疾病的病理, 从心脏病到变性, 癌症16,17,18, 19,20。
考虑到线粒体 ca2 +信号在新陈代谢和细胞死亡中的根本重要性, 并结合线粒体 ca2 +信号影响的生物系统的广泛范围, 评估线粒体 ca2 + 的方法涌入是非常有趣的。毫不奇怪, 测量线粒体 Ca2 +的各种技术和工具已经开发出来。这些方法包括使用荧光 Ca2 +敏感染料21、22和基因编码的 Ca2 +传感器 (如 cameleon 和 aequorin23) 等工具, 24。本文的目的是突出显示可测量线粒体 Ca2 +吸收的不同方法和模型系统。我们提出两种实验方法来评估线粒体 Ca2 +涌入容量。以心肌线粒体为例, 我们详细介绍了一个基于平板阅读器的测量线粒体 ca2 +摄取量的平台, 使用 Ca2 +敏感染料钙 green-5N, 最适合于孤立的组织线粒体14.利用培养的 NIH 3T3 细胞, 我们还描述了一种基于共焦显微成像的检测方法, 用于测量透细胞中线粒体 Ca2 + , 使用 Ca2 +敏感染料 Rhod-2/AM25。

<table>
	<tbody>
		<tr>
			<td>Olympus FV1000 Laser Scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy Neo2 Multimode microplate reader with injectors</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Homogenizer</td>
			<td>Kimble</td>
			<td>886000-0022</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm coverslips</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3628</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Green-5N</td>
			<td>Invitrogen</td>
			<td>C3737</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker green FM</td>
			<td>Invitrogen</td>
			<td>M7514</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhod-2, AM</td>
			<td>Invitrogen</td>
			<td>R1244</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Invitrogen</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P3000MP</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma</td>
			<td>M9546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>EMD Millipore</td>
			<td>8510</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E8145</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>L-malic acid</td>
			<td>Sigma</td>
			<td>M1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher</td>
			<td>BP364-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphocreatine disodium salt</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S7900</td>
			<td></td>
		</tr>
		<tr>
			<td>Ru360</td>
			<td>Calbiochem</td>
			<td>557440</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyses of mitochondrial calcium influx in isolated mitochondria and cultured cells

由线粒体处理的 Ca2 +是调节广泛细胞中生理和病理过程的关键功能。准确测量来自线粒体的 ca2 +的流入和流出的能力对于确定线粒体 ca2 +处理在这些进程中的作用非常重要。在本报告中, 我们提出两种方法来测量线粒体 Ca2 +处理在孤立的线粒体和培养细胞。我们首先详细介绍了一个基于平板阅读器的测量线粒体 ca2 +吸收使用 Ca2 +敏感染料钙 green-5N 的平台。基于平板阅读器的格式绕过了对专用设备的需要, 而钙 green-5N 染料非常适合于从分离的组织线粒体中测量 Ca2 + 。对于我们的应用, 我们描述的测量线粒体 Ca2 +吸收线粒体从小鼠心脏组织分离;但是, 此过程可用于测量线粒体 Ca2 +在与其他组织 (如肝脏、骨骼肌肉和大脑) 隔绝的线粒体中摄取量。其次, 我们描述了以共焦显微镜为基础的检测透细胞线粒体 ca2 +使用 Ca2 +敏感染料 Rhod-2/AM 和成像使用2维激光扫描显微术。此通透协议消除了胞浆染料污染, 允许对线粒体 Ca2 +中的变化进行特定记录。此外, 激光扫描显微镜允许高帧率捕获线粒体 Ca2 +的快速变化, 以应对在外部溶液中应用的各种药物或试剂。此协议可用于测量线粒体 Ca2 +在许多细胞类型中的吸收, 包括主要细胞, 如心肌细胞和神经元, 以及永生化细胞系。

图 1显示了使用基于板阅读器的平台和 Ca2 +染料钙 green-5N, 在孤立的心肌线粒体中线粒体 Ca2 +摄取量测量。在控制条件下 (图 1A), 在含有钙 green-5N 的氯化钾缓冲液中悬浮心肌线粒体, 然后以 CaCl2 (5 ul 0.6 毫米 CaCl2解决方案) 的顺序脉冲来挑战, 150s, 300s, 480s 和690s 时间点。CaCl2添加的时间和数...

Watch this Scientific Journal Video about Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells at JoVE.com

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

线粒体钙在分离线粒体和培养细胞中的流入分析

在这里, 我们提出两个协议, 以测量线粒体 Ca2 +流入孤立的线粒体和培养细胞。对于孤立的线粒体, 我们使用 ca2 +敏感染料钙 green-5N, 详细介绍了一个基于板读取器的 ca2 +导入分析。对于培养细胞, 我们使用 Ca2 +染料 Rhod-2/AM 描述了共焦显微方法。

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

使用碘电磷诱导红血球中的红血球

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

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

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution (Video) | JoVE 

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 ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals (Video) | JoVE 

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Unveiling Mitochondrial Contact Sites and Architectural Insights (Video) | JoVE

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

5-Bromo-2&#39;-Deoxyuridine (BrdU) Labeling of Mitochondrial DNA in HeLa Cells

5-Bromo-2'-Deoxyuridine (BrdU) Labeling of Mitochondrial DNA in HeLa Cells (Video) | JoVE 

HeLa细胞中线粒体DNA的5-溴-2&#39;-脱氧尿苷（BrdU）标记