我们感谢希尔丝廷Elgass博士和莫纳什显微成像技术援助萨拉信条博士和威康信托基金会和医学研究理事会英国的财政支持。 MMcK支持澳大利亚研究理事会未来的奖学金计划（FT120100459），威廉·巴克兰基金会，澳大利亚线粒体病基金会（AMDF），医学研究和莫纳什大学的哈德逊研究所。这项工作是由维多利亚州政府经营性基础设施支持计划的支持。

1.细胞的制备<ol><li>生长10 1cm池培养皿的细胞或75cm 2的烧瓶中补充有5％（体积/体积）胎牛血清（FBS）和1×青霉素/链霉素（P / S培养基[10毫升Dulbecco氏改良的Eagles培养基（DMEM）中）]，在37℃/ 5％CO 2。 </li><li>收获细胞，通过抽吸除去培养基，然后用5ml 1×磷酸盐缓冲盐水（1×PBS）洗涤。通过抽吸除去1×PBS中，然后加入1.5毫升0.25％（重量/体积）胰蛋白酶/ 0.25％（重量/体积）乙二胺四乙酸（EDTA），并在37℃/ 5％CO 2孵育2分钟。点击菜或烧瓶轻轻除去细胞，然后悬浮在5毫升培养基。 </li><li>加入12微升悬浮细胞到血细胞计数器的细胞。 </li><li>板块细胞菜肴或共焦成像腔盖玻片。 注意:这些将有一个合适的塑料或玻璃底已设计用于倒置显微镜的使用。 </li><li>板细胞，使日在〜80％汇合上成像的天来实现。例如，大约2×10 4个 143B骨肉瘤细胞可以接种到成像之前的8孔腔室载玻片一天一个阱。 </li><li>孵育细胞过夜，在37℃/ 5％CO 2，以允许细胞附着和恢复。 </li></ol> 2.缓冲器，用于TMRM和荧光 - 4数字影像<ol><li>制备含有156 mM氯化钠，3毫米氯化钾，2mM的硫酸镁 ，记录溶液（RS） - 1.25毫KH 2 PO 4，10mM的D-葡萄糖，2mM的氯化钙和10mM的HEPES pH值7.35。 RS储存在-20ºC等分。 </li><li>制备细胞内中等（IM）含有6毫摩尔NaCl，130毫摩尔的KCl，7.8毫的MgCl 2，1mM的KH 2 PO 4，0.4mM的氯化钙 ，2毫EGTA，10mM的HEDTA，2毫苹果酸盐，2mM的谷氨酸，2毫ADP和20mM HEPES pH值7.1。苹果酸盐，谷氨酸盐和ADP的的200mM储备溶液可以单独制备，等分并储存在-20℃。这些STocks可以只使用前加入到IM。 </li><li>从商业供应商的 Ca 2+游离Hank氏缓冲盐溶液（HBSS）中，可以得到的预制备。添加乙二醇 - 双（β氨基乙基醚）-N，N，N&#39;，N&#39;-四乙酸（EGTA），以500μM的最终浓度。 </li><li>制备在100％甲醇的10mM四甲甲酯，高氯酸根（TMRM）的贮备溶液。从这种股票使2μm的工作原液在蒸馏水H 2 O </li><li>制备的10mM维拉帕米在100％乙醇中的储备溶液。 </li><li>制备为1mg / ml（重量/体积）的二甲亚砜（DMSO）的Fluo-4乙酰氧基甲基酯（的Fluo-4，AM）的储备溶液。 注:荧光- 4，AM是在结合钙离子表现出增加的荧光钙指标。的Fluo-4是FLUO-3的类似物，由氟取代的两个氯取代基。这导致在488纳米增加的荧光激发和更高的荧光信号水平。该AM酯基团产生的是能够渗透细胞膜不带电的荧光 - 4分子。一旦进入细胞内，在AM酯通过非特异性酯酶裂解，导致在被截留在细胞内的Fluo-4的充电形式。 </li><li>制备在100％乙醇1mM的羰基氰化物对 - trifluoromethoxyphenylhydrazone（FCCP）的贮备溶液。 </li><li>制备的25毫克/毫升的蒸馏水H 2 O储备溶液（重量/体积）毛地黄皂苷</li><li>制备100μM的毒胡萝卜素的DMSO储备溶液。 </li><li>在蒸馏H 2 O制备的40毫氯化钙（氯化钙2）的储备溶液</li></ol> 3. TMRM和荧光 - 4，AM细胞染色<ol><li>用20nM的TMRM制备RS染色溶液，5微克/毫升（重量/体积）的Fluo-4，AM和0.005％表面活性剂，如尼克F-127的RS。 注:这种表面活性剂是一种非离子型多元醇，其促进的Fluo-4，AM的溶解。注:也可加入10μM的维拉帕米到INHIBI吨TMRM出口通过质膜药转运如果它是在细胞类型中表达所分析。 </li><li>通过吸移管除去从细胞培养基，并用100微升1×PBS洗涤。 </li><li>通过吸移管除去1×PBS中并孵育在250微升的RS染色溶液的细胞在室温下45分钟。 注意:一旦的Fluo-4，AM进入细胞中，AM酯通过蜂窝酯酶裂解。在室温下温育抑制酯酶的活性，允许的Fluo-4，AM进入线粒体。 </li><li>通过吸移管除去RS染色液和洗涤细胞在100μl 的 Ca 2+的HBSS以除去过量的Fluo-4，AM和TMRM。 </li><li>用25微克/毫升制备的IM成像溶液（重量/体积）毛地黄皂苷，200nM的TMRM和在IM1μM的毒胡萝卜素。 注:毛地黄皂苷的浓度可能需要进行调整，以确保线粒体内膜不会发生的那透。这可凭经验通过评估强度来确定在不同浓度的毛地黄皂苷的TMRM信号。 </li><li>用移液管除去的 Ca 2+的HBSS，并添加300微升的IM成像溶液到细胞中。离开细胞在室温下平衡10分钟。细胞现在已经准备好与氯化钙补充成像。 </li></ol> 4.细胞成像<ol><li>将菜或在IM成像解决方案细胞腔盖玻片上倒置激光扫描共聚焦显微镜。 </li><li>使用显微镜激光器TMRM和的Fluo-4的激发和发射光谱的设置。例如，使用543纳米的氦氖和473纳米的氩激光线分别以激发TMRM和的Fluo-4。使用大约5％的低激光功率，以最小化对细胞的任何光损伤。 </li><li>设置显微镜扫描图像每25秒。扫描单元10分钟以建立用于TMRM和的Fluo-4的信号的基线读数。 注:荧光 - 4的信号可能是在静息钙concent微弱或无法检测口粮。 </li><li>加入3微升的40mM的CaCl 2储备溶液直接向细胞中加入300μl的IM成像溶液（1:100稀释），并用移液管轻轻混匀。暂停图像扫描同时加入，如果需要混合的CaCl 2。 注:最终的游离钙离子浓度的离子钙离子浓度在IM成像解决方案可以使用软件进行计算，如Maxchelator WEBMAXC扩展（ <a href="http://www.stanford.edu/%7Ecpatton/maxc.html">http://www.stanford.edu/%7Ecpatton/maxc.html</a> ）或螯合剂11。如何计算最终的游离钙离子离子浓度的详细描述钙离子浓度在下面第6条所述。 </li><li>持续约4分钟图像扫描，然后重复氯化钙增加每4分钟，直到所需的TMRM或荧光- 4信号到达，通常在8到10加法。 </li><li>使用吸管，加入1:100稀释1 mM的FCCP的（决赛Concentration 10μM）直接向细胞消散ΔΨ 微米 。图像细胞再5分钟。实验已经结束。 </li></ol> 5.图像分析<ol><li>一旦成像完成后，确定使用图像分析软件的Fluo-4和TMRM信号的强度，对于具有生物格式的插件（下载&#39;bioformats_package.jar"从http://downloads.openmicroscopy.org/bio例如ImageJ的软件 - 格式/并将其保存在"C: Program Files文件的ImageJ 插件"文件夹）。 </li><li>打开ImageJ的影像文件（例如一个.oif文件）。点击选择工具，并选择感兴趣区域（ROI）的区域包含线粒体。使用初始TMRM信号来选择投资回报率。 </li><li>打开"分析&gt;工具&gt;投资回报率经理"，单击"添加"，以包括强度测量选定的投资回报率。多个ROI可以测量单个图像上进行选择。重复上述步骤，直到所有的投资回报率■找被添加到的ROI管理器。 </li><li>点击"更多&gt;多措施"，确保"衡量所有切片"被选中，那是一排每片"被选中，然后点击"确定"，以获得TMRM和荧光 - 4信号的平均荧光强度的表在每个时间点每个ROI。 </li><li>从所有ROI使用数据来计算用于TMRM和的Fluo-4的信号的平均强度和标准偏差。 </li></ol> 6.计算最终游离钙离子离子浓度钙离子浓度 <ol><li>游离钙离子浓度的[Ca 2+]在IM成像解决方案可以使用软件，如Maxchelator WEBMAXC扩展（待定<a href="http://www.stanford.edu/%7Ecpatton/maxc.html">http://www.stanford.edu/%7Ecpatton/maxc.html</a> ）或螯合剂11。例如，设置WEBMAXC变量EXTENDED如下:温度= 24℃，pH值为7。1，离子强度= 0.162，EGTA = 0.002男，HEDTA = 0.01男， 钙 = 0.0004 M和镁离子 = 0.0078 M.这将导致最终的游离钙离子浓度的离子钙离子浓度 62纳米。请注意，这些程序可以不考虑每一个变量，如试剂的纯度，测量和pH测定精度。用于确定游离Ca 2+浓度的最准确的方法是使用经过校准的钙选择性电极。 </li></ol>

<ol>
	<li>Szabadkai, G., 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=Mitochondria:+the+hub+of+cellular+Ca2++signaling.">Mitochondria: the hub of cellular Ca2+ signaling.</a> Physiology. 23, 84-94 (2008).</li><li>Jacobson, J., 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=Interplay+between+mitochondria+and+cellular+calcium+signalling.">Interplay between mitochondria and cellular calcium signalling.</a> Mol. Cell. Biochem. 256-257, 209-218 (2004).</li><li>Bhosale, G., Sharpe, J. A., Sundier, S. Y., 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=Calcium+signaling+as+a+mediator+of+cell+energy+demand+and+a+trigger+to+cell.">Calcium signaling as a mediator of cell energy demand and a trigger to cell.</a> Ann. N. Y. Acad. Sci. 1350, 107-116 (2015).</li><li>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=Mitochondria+calcium-dependent+neuronal+death+and+neurodegenerative+disease.">Mitochondria calcium-dependent neuronal death and neurodegenerative disease.</a> Pflugers Arch. 464, 111-121 (2012).</li><li>Drummond, R. M., Mix, T. C., Tuft, R. A., Walsh, J. V., Fay, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Ca2++homeostasis+during+Ca2++influx+and+Ca2++release+in+gastric+myocytes+from+Bufo+marinus.">Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+ release in gastric myocytes from Bufo marinus.</a> J. Physiol. 522, 375-390 (2000).</li><li>Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., Thomas, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+of+cytosolic+calcium+oscillations+in+the+mitochondria.">Decoding of cytosolic calcium oscillations in the mitochondria.</a> Cell. 82, 415-424 (1995).</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>Pozzan, T., Rudolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+mitochondrial+calcium+in+vivo.">Measurements of mitochondrial calcium in vivo.</a> Biochim. Biophys. Acta. 1787, 1317-1323 (2009).</li><li>Rizzuto, R., Simpson, A. W., Brini, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+of+mitochondrial+Ca2++revealed+by+specifically+targeted+recombinant+aequorin.">Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.</a> Nature. 358, 325-327 (1992).</li><li>Pitter, J. G., Maechler, P., Wollheim, C. B., Spat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+respond+to+Ca2++already+in+the+submicromolar+range:+correlation+with+redox+state.">Mitochondria respond to Ca2+ already in the submicromolar range: correlation with redox state.</a> Cell Calcium. 31, 97-104 (2002).</li><li>Schoenmakers, T. J., Visser, G. J., Flik, G., Theuvenet, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHELATOR:+an+improved+method+for+computing+metal+ion+concentrations+in+physiological+solutions.">CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.</a> BioTechniques. 12, 870-879 (1992).</li><li>McKenzie, 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=Impaired+Cellular+Bioenergetics+Causes+Mitochondrial+Calcium+Handling+Defects+in+MT-ND5+Mutant+Cybrids.">Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids.</a> PLoS One. 11, e0154371 (2016).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat. Rev. Mol. Cell Biol. 1, 11-21 (2000).</li><li>Homolya, L., Hollo, Z., Germann, U. A., Pastan, I., Gottesman, M. M., Sarkadi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+cellular+indicators+are+extruded+by+the+multidrug+resistance+protein.">Fluorescent cellular indicators are extruded by the multidrug resistance protein.</a> J. Biol. Chem. 268, 21493-21496 (1993).</li><li>Fujimoto, K., Chen, Y., Polonsky, K. S., Dorn, G. W. . 2. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cyclophilin+D+and+the+mitochondrial+permeability+transition+enhances+beta-cell+survival+and+prevents+diabetes+in+Pdx1+deficiency.">Targeting cyclophilin D and the mitochondrial permeability transition enhances beta-cell survival and prevents diabetes in Pdx1 deficiency.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 10214-10219 (2010).</li><li>Rao, V. K., Carlson, E. A., Yan, 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=Mitochondrial+permeability+transition+pore+is+a+potential+drug+target+for+neurodegeneration.">Mitochondrial permeability transition pore is a potential drug target for neurodegeneration.</a> Biochim. Biophys. Acta. 1842, 1267-1272 (2014).</li></ol>

The authors declare that they have no competing financial interests.

钙起着在许多细胞过程，包括肌肉收缩，神经元信号传导和细胞增殖13的关键作用。在细胞钙离子浓度的增加往往与能源需求有关，与钙能直接刺激线粒体氧化磷酸化，以提高ATP生成3。因此，重要的是，我们必须有效地监测线粒体钙累积，并且能够以比较如何这个功能是由遗传因素和药剂的影响的能力。 该协议中的关键步骤<p class="jove_cont...

线粒体通过充当本地的 Ca 2+缓冲液1在调节细胞内Ca 2+浓度的一个重要的角色。钙离子通过钙单向转运体进入线粒体，通过跨线粒体内膜（ΔΨ 米 ）2存在电化学梯度驱动的方法。一旦线粒体基质内，钙离子可通过刺激三羧酸循环3三限速脱氢酶激活氧化磷酸化。这种刺激保持ΔΨ 男 ，作为正钙离子越过线粒体内膜进入线粒体基质被暂时消散。如果线粒体内的Ca 2+浓度变得非常高，线粒体通透性转换可以启动，导致ΔΨ 米耗散，cessatio氧化磷酸化的n和细胞死亡的信号传导途径4的诱导。 线粒体在细胞中钙的缓冲空间发挥的重要作用，使线粒体钙的准确的监测非常重要。各种方法已被建立并监控线粒体钙，包括使用基于若丹明染料。一个这样的染料，RHOD-2，AM是在分区到线粒体相当有效测量线粒体的 Ca 2+水平5,6。然而，必须注意用作某些染料将在其他细胞器，如脂质体的积累，或保留在细胞胞质溶胶。然而，下游分析可被用来从那些从线粒体7区分这些信号。 监测线粒体钙另一种技术利用荧光记者构造8 </s<html>了&gt;。这些基因编码的探针的好处是，它们可以通过使用内源性N-末端肽，例如人COX VIII亚基的N-末端的定位信号进行具体靶向线粒体。该系统已被用来产生已经证明了线粒体调查钙信号9是非常有用的线粒体靶向探针水母发光蛋白。这些基因编码的探针的主要缺点是，它们需要被引入到由瞬时表达的细胞（其是不适合某些细胞类型可行，并且能够产生不同的结果），或者通过创建稳定表达系统（这是耗时）。 为了规避上述问题，我们已经开发出一种新的协议，同时测量线粒体钙和ΔΨ 米此协议是基于添加了外源钙到透化的细胞一前述方法S ="外部参照"&gt; 10。我们的协议比其他方法三大优势:首先，我们使用荧光- 4，AM和TMRM监测线粒体钙和ΔΨm，即具有非常独特的光谱特性两种染料;其次，将细胞透化，以使的Fluo-4信号仅检测线粒体的Ca 2+和不钙定位于其它细胞器或细胞胞质溶胶;第三，使用的Fluo-4的检测线粒体的 Ca 2+允许快速和简单的细胞染色，否定存在如果使用遗传编码的探针的任何细胞转染或转化的问题。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>ThermoFisher</td><td>10566016</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>ThermoFisher</td><td>16000044</td><td></td></tr><tr><td>1x phosphate buffered saline (PBS)</td><td>ThermoFisher</td><td>10010023</td><td></td></tr><tr><td>100x penicillin/streptomycin (p/s)</td><td>ThermoFisher</td><td>15140122</td><td></td></tr><tr><td>0.25% Trypsin/0.25% EDTA</td><td>ThermoFisher</td><td>25200056</td><td></td></tr><tr><td>8-well chambered coverslip</td><td>ibidi</td><td>80826</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>793566</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541&amp;nbsp;</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>746452</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>795488</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8270&amp;nbsp;</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>746495</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375&amp;nbsp;</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M2670&amp;nbsp;</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E4378&amp;nbsp;</td><td></td></tr><tr><td>HEDTA</td><td>Sigma-Aldrich</td><td>H8126&amp;nbsp;</td><td></td></tr><tr><td>malate</td><td>Sigma-Aldrich</td><td>M1000</td><td></td></tr><tr><td>glutamate</td><td>Sigma-Aldrich</td><td>G1626</td><td></td></tr><tr><td>ADP</td><td>Sigma-Aldrich</td><td>A5285</td><td></td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt; free Hank&amp;rsquo;s buffered salt solution (HBSS)&amp;nbsp;</td><td>ThermoFisher</td><td>14175-095</td><td></td></tr><tr><td>tetramethylrhodamine, methyl ester, perchlorate (TMRM)</td><td>ThermoFisher</td><td>T668</td><td></td></tr><tr><td>Verapamil</td><td>Sigma-Aldrich</td><td>V4629</td><td></td></tr><tr><td>Fluo-4 acetoxymethyl ester (Fluo-4, AM)</td><td>ThermoFisher</td><td>F14201</td><td></td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>ThermoFisher</td><td>D12345</td><td></td></tr><tr><td>carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td><td>Sigma-Aldrich</td><td>C2920</td><td></td></tr><tr><td>digitonin</td><td>Sigma-Aldrich</td><td>D141</td><td></td></tr><tr><td>thapsigargin</td><td>Sigma-Aldrich</td><td>T9033</td><td></td></tr><tr><td>Pluronic F-127&amp;nbsp;</td><td>ThermoFisher</td><td>P3000MP&amp;nbsp;</td><td></td></tr><tr><td>hemacytometer</td><td>VWR</td><td>631-0925</td><td></td></tr><tr><td>10 cm cell culture dishes</td><td>Corning</td><td>COR430167</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; cell culture flasks</td><td>Corning</td><td>COR430641</td><td></td></tr></tbody></table>

simultaneous measurement of mitochondrial calcium and mitochondrial membrane potential in live cells by fluorescent microscopy

除了在产生的ATP其重要作用，线粒体还充当本地钙（Ca 2+）缓冲器，以紧密地调节细胞内Ca 2+浓度。要做到这一点，线粒体利用电化学势在其内膜（ΔΨ 米 ）隔离的 Ca 2+。 的 Ca 2+的流入线粒体刺激三羧酸循环三个限速脱氢酶，通过氧化磷酸化（OXPHOS）配合增加的电子转移。这种刺激保持ΔΨ 男 ，作为正钙离子越过线粒体内膜进入线粒体基质被暂时消散。 我们在这里描述了使用共聚焦显微镜同时测量线粒体的Ca 2+摄取和ΔΨ 微米活细胞的方法。由透化细胞，线粒体的 Ca 2+能使用荧光钙指示剂的Fluo-4，AM被测定，与使用荧光染料四甲（TMRM）ΔΨ 男 ，甲酯，高氯酸盐测量。本系统的好处是，有荧光染料之间很少光谱重叠，使线粒体的Ca精确测量2+和ΔΨ 米同时。使用顺序除钙等分，线粒体钙的摄取可以被监控，并在其中钙诱导线粒体膜通透性过渡和ΔΨ的损失浓度中号决定。

我们已经使用该协议来检查上143B细胞线粒体的钙12缓冲的增加的能力的MT-ND5突变的影响。在这里所示的例子中，控制143B细胞加载TMRM和的Fluo-4，用毛地黄皂苷透化之前AM。 5分钟成像之后，一个1的8个连续的增补:100稀释的40毫摩尔外源性的CaCl 2被做了，与最终游离Ca 2+离子浓度计算[2+钙。 <p class="jove_content" fo:keep-together.withi...

Watch this Scientific Journal Video about Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy at JoVE.com

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

线粒体可以利用在其内的膜（Δ 膜电势 ）的电化学势螯合钙（Ca 2+），允许它们塑造细胞内的Ca 2+的细胞内信号传导。我们描述了使用荧光染料和共聚焦显微镜同时测量线粒体的Ca 2+摄取和ΔΨ 微米活细胞的方法。

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+ ...

simultaneous-measurement-mitochondrial-calcium-mitochondrial-membrane

Research

JoVE Journal

Biology

18.8K 次观看.  Hudson Institute of Medical Research. 线粒体可以利用在其内的膜（Δ 膜电势 ）的电化学势螯合钙（Ca 2+），允许它们塑造细胞内的Ca 2+的细胞内信号传导。我们描述了使用荧光染料和共聚焦显微镜同时测量线粒体的Ca 2+摄取和ΔΨ 微米活细胞的方法。 

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

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 ...

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

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

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

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

速度更快，高分辨率，MTPA-GFP基于线粒体融合含量的收购多个单元格的动力学数据，在并行使用共聚焦显微镜

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

使用基因编码的电压指示器测量 体内 线粒体膜电位

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

线粒体膜电位和超氧化物水平的定量

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

作者聚焦:在 HeLa 细胞中使用实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...

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

摘要

探索更多视频

此视频中的章节

线粒体钙在分离线粒体和培养细胞中的流入分析

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

测量心肌细胞的快速钙助焊剂

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

速度更快，高分辨率，MTPA-GFP基于线粒体融合含量的收购多个单元格的动力学数据，在并行使用共聚焦显微镜

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

使用基因编码的电压指示器测量体内线粒体膜电位

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

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

摘要

探索更多视频

此视频中的章节

线粒体钙在分离线粒体和培养细胞中的流入分析

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

测量心肌细胞的快速钙助焊剂

线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验

速度更快，高分辨率，MTPA-GFP基于线粒体融合含量的收购多个单元格的动力学数据，在并行使用共聚焦显微镜

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

使用基因编码的电压指示器测量 体内 线粒体膜电位

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

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

使用基因编码的电压指示器测量体内线粒体膜电位