Name: analyses of mitochondrial calcium influx in isolated mitochondria and cultured cells
Uploaded: 2018-04-27T13:30:00.000+00:00
Duration: 8 min 29 s
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 '-四乙酸四酸 (EGTA), pH 值调整为7。4与 KOH。通过0.22 微米的过滤器消毒, 并存储在4摄氏度。在使用前, 请确保 MS EGTA 缓冲器预冷至4摄氏度。</li>
	<li>准备100毫升氯化钾缓冲器: 125 毫米氯化钾, 20 毫米 HEPES, 1 毫米2PO4, 2 毫米氯化镁2, 40 微米 EGTA 和 pH 值调整为7.2 与 KOH。把它储存在室温下。</li>
	<li>在二甲基亚砜 (亚砜) 上制备1毫米钙 green-5N。钙 green-5N 的库存可以 aliquotted, 并储存在-20 摄氏度。</li>
	<li>
为 Ca2 +吸收准备基板。
	<ol>
<li>准备1米丙酮酸钠, pH 值7.4。储存在整除数-20 摄氏度。</li>
		<li>准备500毫米苹果酸, pH 值7.4。储存在整除数-20 摄氏度。</li>
	</ol>
</li>
</ol>2. 心肌线粒体的分离
<ol><li>根据制度标准弄死老鼠。 注: 对于我们在制度上批准的安乐死方法, 小鼠被 isofluorane 吸入并因颈椎脱位而被麻醉。</li>
	<li>通过打开胸腔, 沿肋骨两侧切开, 心脏两侧, 切断隔膜, 切除心脏组织来收集心脏。 注意: 在这个协议中, 线粒体隔离是从一个成年小鼠 (大约120毫克) 收集的整个心脏进行的, 这应该足够3-4 实验。对于线粒体与其他富含线粒体的组织 (如肝脏) 的分离, 可根据所描述的协议使用多达200毫克的组织。</li>
	<li>彻底冲洗组织在25毫升的冰冷1x 磷酸盐缓冲盐水 (PBS), 确保所有的血液被挤出心室。 注: 当液体从心脏挤出时, 心脏会充分冲洗。</li>
	<li>用一把锋利的剪刀把心切成5毫升的冰冷 1x PBS 中的小块。</li>
	<li>丢弃 PBS 并将碎心组织转移到预冷的7毫升玻璃-聚四氟乙烯 dounce 均质机, 其间隙为0.10 到0.15 毫米。</li>
	<li>加入5毫升冰冷 EGTA 缓冲液, 融汇样品, 直到组织片不再可见 (大约11冲程)。 注意: 不要过度融汇组织, 因为目标是获得完整和功能的线粒体。</li>
	<li>将匀浆转移到15毫升管上。</li>
	<li>离心匀浆在 600 x g 在4°c 为5分钟对球团核和未完整的细胞。</li>
	<li>将上清液转移到新鲜的15毫升管, 并将其离心在 1万 x g 处, 4 摄氏度, 以颗粒线粒体为10分钟。</li>
	<li>去掉上清, 将线粒体颗粒放在冰上。</li>
	<li>用冰冷的 MS-EGTA 缓冲液清洗线粒体颗粒两次。每个洗涤, 并用重悬线粒体颗粒在5毫升的 MS EGTA 缓冲, 离心它在 1万 x 克4摄氏度10分钟, 并丢弃上清。</li>
	<li>最后洗完后, 弃上清, 并用重悬 100 ul EGTA 缓冲液中的线粒体。把线粒体放在冰上。 注: 线粒体应用于实验1小时以内。</li>
	<li>使用布拉德福德蛋白测定法测定线粒体蛋白浓度26。</li>
</ol>3. 基于平板阅读器的线粒体钙摄取量测量
注: 在这里, 它被描述的协议, 以分析线粒体 Ca2 +吸收使用一个多模版读卡器装有注射器。任何能读取钙 green-5N 荧光 (506/532 nm 的激发/发射) 的平板阅读器, 都可以使用自动的试剂注射器来保持从光中保护的反应。
<ol><li>程序板阅读器执行动态读取钙 green-5N 荧光与测量每秒为 1000 s 的总化验时间. 另外, 程序的试剂注射器分配 5 ul 的 CaCl2解决方案在三十年代, 150s, 300s、480s 和690s 时间点。 注意: CaCl2注入的时间是用户定义的, 可以根据实验需要进行调整。</li>
	<li>将使用 CaCl2解决方案的试剂注入器作为质数。 注意: CaCl2解决方案的集中度是用户定义的, 可以在随后的运行中进行调整, 以滴定触发 MPTP 打开所需的 Ca2 +的数量。</li>
	<li>将200微克的线粒体添加到一个96井板的单个井中。</li>
	<li>在井中加入适量的氯化钾缓冲液, 使线粒体和氯化钾缓冲器的总容积达 197 ul。</li>
	<li>添加 1 ul 1 M 丙酮酸, 1 ul 的500毫米苹果的线粒体混合物。吸管轻轻地混合, 并在室温下孵化线粒体与基质2分钟, 使线粒体变得精力充沛。</li>
	<li>添加 1 ul 1 毫米钙 green-5N 库存。轻轻地混合吹打。 注: 在添加钙 green-5N 染料后, 保护反应不受光照。</li>
	<li>启动预编程的动力学协议和监测钙 green-5N 荧光。</li>
</ol>4. 试剂和溶液
注: 第二部分是培养细胞中线粒体 Ca2 +共焦成像的实验程序
<ol><li>使 Tyrode 的溶液 (130 毫米氯化钠, 4 毫米氯化钾, 2 毫米 CaCl2, 1 毫米氯化镁2, 10 毫米葡萄糖, 10 毫米 HEPES, pH 7.2 与 KOH)。储存在4摄氏度。使用前请将其预热至室温。</li>
	<li>准备洗涤液 (100 毫米醋酸钾, 15 毫米氯化钾, 5 毫米的2PO4, 5 毫米毫克 ATP, 0.35 毫米 EGTA, 0.12 毫米 CaCl2, 0.75 毫米氯化镁2, 10 毫米磷酸肌酸, 10 毫米 HEPES, pH 7.2 与 KOH)。储存在4摄氏度。使用前请将其预热至室温。</li>
	<li>准备通透溶液, 在洗涤液中含有0.005% 皂苷。每天准备新鲜的。</li>
	<li>准备 0 Ca2 +内部解决方案 (100 毫米醋酸钾, 15 毫米氯化钾, 0.35 毫米 EGTA, 0.75 毫米氯化镁2, 10 毫米 HEPES, pH 值调整为7.2 与 KOH)。储存在4摄氏度。使用前请将其预热至室温。</li>
	<li>准备包含 Ca2 +的内部解决方案。将 CaCl2添加到上面的内部解决方案, 以达到所需的可用 Ca2 +的浓度。使用 MaxChelator 程序 (maxchelator.stanford.edu) 计算要添加的 CaCl2的数量。</li>
	<li>准备1毫米 Rhod-2/AM 股票在亚砜和存储它在-20 °c, 直到使用。</li>
	<li>准备1毫米 MitoTracker 绿色股票在亚砜和存储它在-20 °c, 直到使用。</li>
</ol>5. 用于成像的电镀细胞
<ol><li>洗涤 22 x 22 cm2玻璃盖玻片与100% 乙醇并且允许盖玻片空气干燥。</li>
	<li>将玻璃盖玻片放入6井组织培养皿的单个水井中。 注: 盖玻片可涂覆层粘连蛋白、聚 l-赖氨酸或类似基质, 促进细胞附着。</li>
	<li>胰蛋白酶处理和平板细胞上的盖玻片瞄准大约70% 汇合日的成像。</li>
</ol>6. 用 Rhod-2/AM 和 MitoTracker 绿色装载细胞
<ol><li>准备 Rhod-2/AM-MitoTracker 绿色工作解决方案。添加 20 ul 的 Rhod-2/AM 1 毫米股票, 0.2 ul MitoTracker 绿色1毫米股票, 2.5 ul 的 20% pluronic F-127 到1毫升 Tyrode 的解决方案。Rhod-2 在工作溶液中的最终浓度为20µM, MitoTracker 绿色的最终浓度为200纳米。</li>
	<li>从盖玻片中轻轻移除生长介质。 注: 在染料溶液添加之前, 洗涤步骤是不必要的。</li>
	<li>将 Rhod-2/AM-MitoTracker 绿色溶液以滴状的方式添加到盖玻片上, 直到盖玻片被覆盖 (每盖玻片大约3-4 滴)。</li>
	<li>在室温保护下, 在室温下孵化30分钟, 使细胞与染料一起加载。</li>
	<li>酯化 Rhod-2/AM。轻轻地去除 Rhod-2/AM-MitoTracker 绿色溶液, 用新鲜室温 Tyrode 的溶液代替它, 在室温下将其孵化30分钟以保护光线。</li>
</ol>7. 线粒体 Rhod-2/AM 和 MitoTracker 绿荧光的共焦成像
<ol><li>将盖玻片转移到显微镜成像室, 用洗涤液填充腔室。</li>
	<li>在40X 的相位对比观察单元格的设置下, 调整焦点, 直到单元格可见并聚焦。</li>
	<li>
Permeabilize Rhod-2/AM-MitoTracker 绿色负载细胞的等离子膜去除细胞质局部 Rhod-2, 同时保留线粒体局部染料。
	<ol>
<li>从盖玻片中取出洗涤液。</li>
		<li>用通透解决方案替换约1分钟。</li>
		<li>在整个通透过程中直观地监测细胞膜形态学。透细胞会形成粗糙的表面。</li>
		<li>通透完成后, 立即删除通透解决方案, 并将其替换为 0 Ca2 +内部解决方案。</li>
	</ol>
</li>
	<li>同时图像 Rhod-2 荧光 (激发使用 559 nm 激光线和发射波长之间的 575-675 nm) 和 MitoTracker 绿色荧光 (激发使用488nm 激光线和发射波长之间收集505-525 毫微米)。关注透细胞, 显示清晰的定位 Rhod-2 和 MitoTracker 绿色。</li>
	<li>减少显微镜激光和增益设置, 使线粒体 Rhod-2 荧光暗淡, 只是可见。</li>
	<li>选择显微镜设置以在适当的帧速率和时间过程中获取特定应用程序的2维扫描。建议至少30帧/秒的帧速率来准确捕获线粒体 Ca2 +中变化的动力学。</li>
	<li>卸下 0 Ca2 +内部解决方案, 而不干扰单元格或显微镜焦点。</li>
	<li>开始图像采集。</li>
	<li>添加 Ca2 +-充满内部解决方案在十年代的时间点, 无论是手动或与灌注系统。</li>
	<li>选择感兴趣的区域 (ROIs), 包括图像采集软件中线粒体 Rhod-2 和 MitoTracker 绿色信号之间的定位区域。 注: 每一个记录的时间点的任意荧光 (F) 值都是为每个 ROI 获取的。这些值是背景减去并规范化为初始荧光 (在 Ca2 +加法之前;f0), 并绘制为 f/f0随着时间的推移, 数据呈现和量化的幅度变化的线粒体 Ca2 +。</li>
</ol>

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N., Blatter, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+cardiac+mitochondria.">Calcium signaling in cardiac mitochondria.</a> J Mol Cell Cardiol. 58, 125-133 (2013).</li><li>Florea, S. M., Blatter, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+for+the+regulation+of+cardiac+alternans.">The role of mitochondria for the regulation of cardiac alternans.</a> Front Physiol. 1, 141 (2010).</li><li>Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+calcium+signaling+using+genetically+targetable+fluorescent+indicators.">Measuring calcium signaling using genetically targetable fluorescent indicators.</a> Nat Protoc. 1 (3), 1057-1065 (2006).</li><li>Bonora, M., 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=Subcellular+calcium+measurements+in+mammalian+cells+using+jellyfish+photoprotein+aequorin-based+probes.">Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes.</a> Nat Protoc. 8 (11), 2105-2118 (2013).</li><li>Zima, A. V., Kockskamper, J., Mejia-Alvarez, R., Blatter, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyruvate+modulates+cardiac+sarcoplasmic+reticulum+Ca2++release+in+rats+via+mitochondria-dependent+and+-independent+mechanisms.">Pyruvate modulates cardiac sarcoplasmic reticulum Ca2+ release in rats via mitochondria-dependent and -independent mechanisms.</a> J Physiol. 550 (Pt 3), 765-783 (2003).</li><li>Kruger, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bradford+method+for+protein+quantitation.">The Bradford method for protein quantitation.</a> Methods Mol Biol. 32, 9-15 (1994).</li><li>Zazueta, C., Sosa-Torres, M. E., Correa, F., Garza-Ortiz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+properties+of+ruthenium+amine+complexes+on+mitochondrial+calcium+uptake.">Inhibitory properties of ruthenium amine complexes on mitochondrial calcium uptake.</a> J Bioenerg Biomembr. 31 (6), 551-557 (1999).</li><li>Davidson, S. M., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mitochondrial+calcium+signalling+with+fluorescent+probes+and+single+or+two+photon+confocal+microscopy.">Imaging mitochondrial calcium signalling with fluorescent probes and single or two photon confocal microscopy.</a> Methods Mol Biol. 810, 219-234 (2012).</li><li>Matlib, M. A., 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=Oxygen-bridged+dinuclear+ruthenium+amine+complex+specifically+inhibits+Ca2++uptake+into+mitochondria+in+vitro+and+in+situ+in+single+cardiac+myocytes.">Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes.</a> J Biol Chem. 273 (17), 10223-10231 (1998).</li><li>Chamberlain, B. K., Volpe, P., Fleischer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+calcium-induced+calcium+release+from+purified+cardiac+sarcoplasmic+reticulum+vesicles.">Inhibition of calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles.</a> J Biol Chem. 259 (12), 7547-7553 (1984).</li></ol>

作者没有披露报告。

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&amp;prime;-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 ...

analyses-mitochondrial-calcium-influx-isolated-mitochondria-cultured

Research

JoVE Journal

Biochemistry

13.7K 次观看.  Emory University School of Medicine. 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。

视频: Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

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

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

生物学

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

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

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

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验

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

神经科学

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

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

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

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 ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.

钙内流测定法测量培养细胞中线粒体钙摄取

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
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JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

线粒体钙摄取测定:一种基于读板器的测量离体线粒体钙摄取的方法

Biochemical assays

Determination of the Calcium Retention Capacity of Isolated Mitochondria

Source: Li, W., et. al. <a href="https://app.jove.com/v/56236">Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

Source: Li, W., et. al. Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay. J. Vis. Exp. (2018)
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线粒体钙在分离线粒体和培养细胞中的流入分析

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