Name: mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay
Uploaded: 2018-05-01T12:30:00.000+00:00
Duration: 5 min 53 s
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厘米细胞培养皿中用大约1毫升的冰冷 PBS 冲洗细胞3次。</li>
	<li>在10厘米细胞培养皿中加入新的1毫升冰冷 PBS, 将黏附细胞刮成新的1.5 毫升管。离心机在 800 x g 10 分钟在4摄氏度。</li>
	<li>在离心过程中, 准备5毫升线粒体隔离缓冲 (250 毫米蔗糖, 10 毫米 HEPES (4-(2-羟乙基)-1-哌嗪磺酸), 10 毫米氯化钾, 1.5 毫米氯化镁2, 1 毫米 EDTA) 补充 50 ul 蛋白酶抑制剂库存溶液 (100x 浓度; 1 无 EDTA 片剂溶于500µL 的 ddH2O)。</li>
	<li>取出上清液, 并用重悬1毫升线粒体隔离缓冲颗粒。在冰上孵化1.5 毫升管30分钟。</li>
	<li>溶解在冰上手动向上和向下的玻璃均质机 (15-25 冲程) 悬浮细胞。 注意: 确保细胞均匀时没有气泡。用显微镜观察完整的细胞和裸露的细胞核, 并确认 > 90% 细胞破损发生。</li>
	<li>将匀浆转化为1.5 毫升管, 离心机在 1000 x g 处, 5 分钟到4摄氏度, 去除碎片 (细胞核和剩余完好的细胞)。</li>
	<li>将上清液转移到新的1.5 毫升管和离心机在 1000 x g 5 分钟4°c。</li>
	<li>将上清液转移到新管和离心机在 1.4万 x g 15 分钟4°c。</li>
	<li>并用重悬1毫升线粒体隔离缓冲液去除含有线粒体的颗粒。</li>
	<li>离心溶液为15分钟在 1.4万 x g 4 °c 沉淀线粒体。</li>
	<li>所产生的线粒体颗粒必须保持在冰和悬浮 50-100 ul 氯化钾介质 (125 毫米氯化钾, 2 毫米 K2HPO4, 1 毫米氯化镁2, 20 毫米 HEPES, 5 毫米谷氨酸, 5 毫米苹果酸, 2 微米鱼藤酮, pH 7.4) 根据颗粒体积前任何驴哎。</li>
	<li>使用5的线粒体溶液 ul 测定线粒体蛋白浓度通过标准的评估测试, 测量 OD562 使用一个板读取器13。</li>
</ol>2. 线粒体 Ca2 +保留容量的测定
<ol><li>准备氯化钾介质, 并在实验前将 Ca2 +结合绿色荧光染料添加到最终浓度为0.5 微米。</li>
	<li>在含0.5 微米 Ca2 +的氯化钾介质中转移1毫升的分离线粒体 (0.4 毫克/毫升), 将绿色荧光染料结合到每个6井板中。</li>
	<li>孵化线粒体和 Ca2 +-结合绿色荧光染料在6井板室温保护, 从环境光1分钟. 添加 4 ul 整除数20毫米 CaCl 2 解决方案 (20 毫米 CaCl 2, 127 毫米氯化钾, 1 毫米氯化镁 2 , 20 毫米 HEPES, 5 毫米谷氨酸, 5 毫米苹果酸, pH 7.4) 到每个6井板井使用自动分配设置引入 200 nmol Ca2 +/镁线粒体蛋白。</li>
	<li>使用荧光光谱仪监测每3秒的荧光变化为2分钟, 激发波长为 506 nm, 发射波长为 531 nm。在软件控制的读数 (图 1) 之间, 板上的震动为 600 rpm 3 秒。</li>
	<li>添加一个额外的 4 ul 整除20毫米 CaCl2解决方案, 每个井和监测荧光变化每3秒为2分钟. 重复此步骤, 直到荧光随时间增加。 注意: 通过增加的记录荧光指示的添加的 Ca2 + , 线粒体面临挑战。当 mPTP 打开时, 荧光随时间增加。</li>
	<li>解释添加的 ca2 +的总金额, 直到 mPTP 作为 Ca2 +保留容量打开为止。</li>
</ol>3. 钙2 +诱导的线粒体肿胀的测定
<ol><li>在实验前准备氯化钾介质。<ol>
<li>将氯化钾介质中1毫升的分离线粒体 (0.4 毫克/毫升) 转移到每个6井板中。</li>
	</ol>
</li>
	<li>添加 10 ul 20 毫米 CaCl2水溶液 (500 nmol/镁线粒体蛋白) 成线粒体蛋白, 引发线粒体肿胀。</li>
	<li>记录由软件控制的微板块读取器上每3秒 540 nm 的吸光度降低10分钟 (图2)。荧光的变化是由溶质流入线粒体内膜引起的。 注: 540 nm 的吸收量减少, 相当于肿胀的线粒体。如果阅读器没有观察到吸收的减少, 可以采用更长的阅读间隔或阅读时间。</li>
</ol>

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太阳监督实验。李伟进行了实验。陈张和 x. 孙写了这篇论文。

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化的线粒体可以通过1.0 米/1.5M 不连续蔗糖梯度获得。在活体细胞中线粒体的生物学特性更像是体内, 而不是线粒体中的介质成分浓度。线粒体 Ca2 +保留容量或 Ca2 +触发的线粒体肿胀检测是在通电条件下确定的。为确定功能性线粒体, 5 毫米琥珀酸可以添加为呼吸道基质, 而不是谷氨酸和苹果酸6。
ca2 +-结合绿色荧光染料 (绑定 ca2 +具有 4.29 @ 0.67 毫米的亲和性), 更适合于测量 Ca2 +的 micromolar 浓度, 而不是更高亲和染料 (如 fura-2 7)。添加 ca2 +绑定绿色荧光染料后, 孵化必须小于 240s. 连续添加的 ca2 + (不同于 25-200 nmol) 被注射在 1-3 分钟间隔。校准曲线显示出线粒体无法封存的 ca2 +的浓度, 表明 ca2 +的脉冲的线粒体摄取量。否则, ca2 +结合绿色荧光染料也用于钙信号调查, 如胞内游离 Ca2 +浓度测量, 以下 ca2 +流入和释放, 多光子激发活体组织中 Ca2 +的成像。
为确定 Ca2 +诱导的线粒体肿胀, 也可以诱发肿胀, 增加200毫米 ATR 和 CaCl2。作为一个控制, 线粒体可以孵化1毫米 CsA 在测量前5分钟, 这抑制了 mPTP 开放。先前的研究显示, 吸收的校准曲线是肿胀线粒体的百分比的函数14。据报道, 1 毫米 CsA, 0.1 毫米钌红, 和相同体积的新鲜线粒体 (0.5 毫克/毫升) 可以增加的反应时, 肿胀是完整的。由于 CsA 和钌红可以抑制新鲜添加的线粒体中的 mPTP 开放, 校准曲线表明肿胀的线粒体和非肿胀的线粒体15。在 ca2 +过载的情况下, 线粒体肿胀的协议也是对 ca2 +诱导的 mPTP 开放的有效和直接测量。
自从50年前, 从哺乳动物和其他更高脊椎动物的线粒体中发现了 Ca2 +的传输, 对线粒体钙外流和流入的机制和功能进行了大量的研究。线粒体 ca2 +吸收机制包含线粒体 ca2 + uniporter, 快速模式或 RaM, 和兰尼碱受体 (mRyR) 16.我们上面描述的化验结果可以简单有效地评估与 Ca2 +相关的线粒体功能。

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>Ca&lt;sup&gt;2+&lt;/sup&gt;-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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>00457</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;rsquo;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 mPTP 打开被解释为 Ca2 +保留容量。我们的数据显示, BKA 治疗的线粒体 Ca2 +保留容量远高于 ATR 组。这些数据证实了对线粒体 Ca2 +保留容量 (图 3) 的适当测量。
Ca2 +引起的线粒体肿胀的代表性结果: 在添加 CaCl2后的10分钟内, 结果被表达为 540 nm 的吸收率和初始吸光度的百分比变化。由于添加了 Ca2 + (500 nmol/毫克线粒体蛋白), 在 540 nm 监测到的吸收量减少表明线粒体肿胀。BKA 或 atr 的治疗, 校准曲线显示轻微或急剧下降与初始吸光度相比, 表明 BKA 可以抑制 mPTP 开放, 而 ATR 可以促进 mPTP 打开 (图 4)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56236/56236fig1.jpg"> 图 1: 线粒体 Ca 的软件设置2 +保留容量检测.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56236/56236fig2.jpg"> 图 2: 线粒体 Ca 的软件设置2 +触发的线粒体肿胀检测.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56236/56236fig3.jpg"> 图 3: 对 Ca2 +保留容量的测量.用 ca2 +结合绿色荧光染料, 在存在5微米 bongkrekate (BKA)、1微米苍术苷二 (ATR) 或空白控制 (fluorometrically) 的情况下, 测量了分离线粒体中的超线粒体Ca 2 +。使用 BKA、ATR 和微板块的独立线粒体对 Ca2 + 保留的跟踪量以 506 nm (Ex) 和 531 nm (Em) 为单位进行测量。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56236/56236fig4.jpg"> 图 4: 检测 Ca2 +引起的线粒体肿胀.在5微米 bongkrekate (BKA)、1微米苍术苷二 (ATR) 或空白控制 (SH-SY5Y) 存在下,Ca2 + 诱导的线粒体肿胀是以 540 nm 的初始吸光度的百分比降低的校准曲线表示的。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

mitochondrial-ca2-retention-capacity-assay-ca2-triggered

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

11.1K 次观看.  Qilu Hospital of Shandong University. 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。

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

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

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

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

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

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

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in 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 ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

线粒体钙在分离线粒体和培养细胞中的流入分析

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

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

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 ...

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.remotexs.ntu.edu.sg/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).
This video ...

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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)
This video ...

线粒体 Ca^{2 +}保留容量检测和 Ca^{2 +}触发的线粒体肿胀试验

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