我们感谢劳瑞Destroismaisons和出色的技术支持莎拉Peyrard和Dr.Alexandre宝勒访问的流式细胞仪。我们也想感谢蒂莫西·米勒博士，他的贡献就从筹备去除髓鞘。这项工作是由卫生研究院（CIHR）神经肌肉研究合作伙伴，加拿大创新基金会，加拿大ALS协会，基金会弗里克ALS研究，密友基金会和全宗德拉RECHERCHE桑特连接魁北克（CVV）加拿大研究院的支持。无论是CVV和NA是研究学者的全宗德拉RECHERCHE桑特连接魁北克与CIHR新的调查。 SP被蒂姆·诺埃尔助学金来自加拿大的ALS协会的支持。

在这项研究中使用的动物严格按照协议（N08001CVsr）批准的中心德RECHERCHE杜中心医院DE L&#39;蒙特利尔大学（CRCHUM）机构委员会动物保护的它遵循概述了加拿大国家标准进行治疗理事会动物护理（CCAC）。 准备执行该协议（ 表1）所需要的所有试剂。关于设备，耗材及供应商的所有其他细节可以在材料清单上找到。 <img alt="表1" fo:content-width="5in" src="/files/ftp_upload/51887/51887table1.jpg" width="500" /> 表1缓冲区组成。 1，收集大鼠脊髓<ol><li>深深地麻醉大鼠（只SD）与4％异氟烷。在捏个的验证anaesthetization因缺乏反射ê前爪。通过断头台安乐死的大鼠断头。安乐死该方法优于其他，这可能会歪曲脊髓。 </li><li>切开背部皮肤，以暴露脊柱。切骨剪略高于骨盆脊柱。可视化的脊柱开幕。 </li><li>将10毫升注射器，用200微升枪头（通过在火焰稍微融化附后），充满磷酸盐缓冲液（PBS），到脊柱。 </li><li>通过施加的压力的培养基中量到柱塞冲洗出脊髓。 </li><li>如果任何血液存在于脊髓，在进行下一步骤之前，漂洗用PBS。 注:此方法也被证实为脑和肝。如果包括这些组织中，收集半从安乐死的大鼠的脑和一片肝相等的权重至脊髓。所有其他步骤保持一致。 </li></ol> 2，隔离脊髓麻省理工学院ochondria（改编自范德维尔德等。4） <ol><li>收集整个完整的脊髓和地点与5卷（〜3.25毫升）匀浆缓冲液（HB）5毫升玻璃匀浆器。对于分离线粒体的最佳恢复，冰或冷室执行所有步骤。用手均匀的组织，直到没有大块的组织仍然存在，大约8招。置于匀浆中的两（2毫升），三（1.7毫升）的微量离心管中。离心1300 XG在4℃的台式离心10分钟。 </li><li>回收上清液并置于5ml的超离心管。加入750微升（约0.5倍体积）HB到含有颗粒状的离心管中，轻轻地重悬沉淀。重复离心和重悬步骤两次以上。池中的所有上清液（S1A和S1B）到同一个5毫升超速上面管。此步骤用于去除小碎片。 </li><li>汇集离心机使用S1配备了吊桶式腐超速离心机或离心在17000×g离心15分钟，在4℃。 </li><li>保留上清液（S2），用于进一步的处理，如果胞浆部分感兴趣（例如，用于Western印迹分析）。重悬沉淀（P2）的粗线粒体级分，在4毫升HB +的50mM的KCl。离心17000×g离心4℃15分钟的水平转头。弃上清，轻轻悬浮在800微升乙肝颗粒（P3）。 注:线粒体是用HB +的50mM的KCl，以除去任何非特异性线粒体相关的污染物。 </li><li>在新的5毫升超速离心管，加入重悬沉淀（P3），正好800微升。 </li><li>向该试管中加入200微升碘克沙醇（密度梯度介质），从而产生12％的碘克沙醇的终浓度。轻轻的，但通过彻底吸取了P1000的混合管的内容。此外碘克沙醇的第一，然后再悬浮沉淀（P3）可以是优选的，以便充分混合。离心机的ULTracentrifuge在4℃下配备吊桶式转子中以17000×g离心15分钟。 </li><li>注:肝不包含髓鞘，因此，如果仅在肝脏正在处理这一步骤不是必需的。但是，如果肝并发中枢神经系统线粒体进行处理，建议同等对待所有的组织。 </li><li>吸髓磷脂的层在该管的顶部，小心地取出并丢弃上清液。球团可能松动。悬浮在4ml HB沉淀。再次离心在17000 XG在4℃下10分钟。弃上清，悬浮在4毫升HB沉淀。重复离心，去除上清。 </li><li>悬浮最终沉淀（P7）在100-200微升HB和转移到1.7 ml离心管。此示例包含分离线粒体。 </li><li>继续进行蛋白定量。稀释样品并在2％十二烷基硫酸钠（SDS）的标准曲线，以确保线粒体杜里充分溶吴蛋白定量。 </li></ol> 3，免疫标记分离线粒体的流式细胞仪<ol><li>对于每一个染色组合进行测试，吸管25微克分离线粒体到1.7 ml离心管。包括在每个实验未染色的样品;并为每个抗体，一定要包括相应的​​同型对照样本。 </li><li>离心机在17000×g离心2分钟，在4℃下，在台式离心。 </li><li>除去上清，悬浮分离的线粒体在50μl线粒体缓冲液（M缓冲液）于4℃（封闭步骤）补充有10％无脂肪酸BSA的15分钟。 注:在标签，在冰箱中4℃进行孵化。 </li><li>第一抗体（兔抗-Mfn2的，每毫升含20微克）加入到管中，并温育30分钟，在4℃下。 注:用滴定法确定每个抗体的最佳浓度经验。由于变异浓度和/或对urity，不同批次来自同一制造商的同一抗体可能导致不同的结果;因此滴定需要为每个新的大量抗体。 </li><li>在4℃离心17000×g离心2分钟:洗出未结合的抗体。除去上清液，并轻轻悬浮于200μl缓冲液M的颗粒。离心机在5185 XG在4℃下2分钟。除去上清液，重悬在50微升M缓冲沉淀。 </li><li>第二抗体（驴抗兔IgG藻红蛋白（PE），0.5微克每毫升）添加至试管孵育样品30分钟，在4℃，避光。 </li><li>在4℃离心17000×g离心2分钟:洗出未结合的二级抗体。弃上清，重悬在200微升M缓冲沉淀。离心机在5185 XG在4℃下2分钟。除去上清液，重悬在500μlM缓冲沉淀。 </li><li>为确保活动其实都是线粒体，染色分离线粒体用线粒体特异性荧光染料15分钟，在室温下，避光。如果其他功能参数（线粒体跨膜电位或过氧化产物）染色的需要，继续执行步骤4。如果没有，请继续收购步骤5。 注意:验证该第二抗体的发射光谱是与该功能性染料的相容是很重要的。例如，如果验证线粒体纯度用市售染料和类似FITC和跨膜电位与四甲甲酯（TMRM）的光谱特性，一个可行的二级抗体将别藻蓝蛋白（APC:实施例650纳米/ EM 660纳米）。添加补偿控制， 即免疫标记或染色用一个荧光基团，在适用的样本。 </li><li>转移到适合装载流式细胞仪的管子。 （为方便样本量小，微量滴定管放置在3毫升流式细胞仪管。）保持样品在冰上，并立即着手流式细胞仪的采集。 </li></ol> 4，含量测定线粒体跨膜电位与线粒体超氧化物生成流式细胞仪<ol><li>验证分离的线粒体通过用100nM TMRM（实施例548纳米/ EM 574纳米）5，染色在步骤3.8，为15分钟，在室温下，避光有一个完整的跨膜电位。对样品和种群，使用TMRM（1〜30纳米）的下部/非淬火浓度之间的跨膜电位的比较，可以更适当6。 <ol><li>至于TMRM染色，染色分离线粒体与100纳米TMRM在100微米羰基氰氯苯肼（CCCP）的存在，线粒体解耦联，将线粒体去极化控制。 CCCP的去极化的线粒体所需的浓度可以小于，如果使用较低的染料的浓度。 </li></ol></li><li>验证分离线粒体产生线粒体超氧由圣用合适的线粒体超氧化物指示器7癌宁，也是在步骤3.8，为15分钟，在室温，避光。 <ol><li>作为线粒体超氧化物的产生，色斑分离线粒体用染料在10μM抗霉素A，呼吸链，这将增加线粒体超氧化物生成的复合物III抑制剂的存在下的控制。 </li></ol></li></ol> 5，采集免疫标记与分析分离线粒体流式细胞仪<ol><li>仪器设置:从线性电压切换登录方式，以利分离线粒体，并设置电压的分析（FSC:450; SSC:250）。确保事件被收集在FSC-A（面积）模式以及FSC-W（宽度）和FSC-H（高度），以便能够排除双峰（ 即，两个事件，通过检测器传递的同时）在分析后的数据集合。事件的张数设定为被收集到100,000。获得补偿控制（如适用）。 </li><li>数据采集​​:数据采集前，避免涡旋样本。代替通过轻轻敲击管混匀。最初收集在低压活动，浇注过程中。门上的总人口。调整直方图的电压相应地，通常是未染色的样品的峰值将对应于第二个十年（10 2）。一旦门被建立，并且样品正在处理中，压力可切换到高。 </li><li>分析:通过绘制FSC-W与FSC的可视化双峰。确定汗衫及双峰。门上的汗衫。通过门控上都与线粒体选择性染料染色阳性事件选择线粒体人口。 </li><li>确定由同型对照背景标记。使用同种型对照样品，测定线粒体人口标记阳性Mfn2的抗体的百分比。 </li></ol>

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作者什么都没有透露。

这是越来越明显，线粒体是关键球员在正常生理和疾病。虽然免疫可以确定哪些蛋白质在线粒体或在线粒体表面发现了在一定的条件下，对​​整个人口的平均这个方法报告。此方法不能产生约线粒体亚群或亚群的相对丰度信息。虽然先前已假设所有线粒体创建相等，则该字段被越来越多地认识到在小区内的线粒体在形态和/或函数10方面的广泛变化。 荧光显微镜方法也考虑到线粒体异质性。然而，这种类型的数据的量化是劳动密集型的。此外，该方法更适合于使用培养细胞的研究，如线粒体体内的标签/原位是困难的，由于exces西伯数量目前线粒体，使个体的细胞器的分化本来就很难。在大多数免疫细胞化学实验的协议，也无法同时标记线粒体外膜蛋白和评估线粒体功能由于所需的抗体标记的细胞透化步骤。当前的方法适用于分离的线粒体中，并且因此不需要透化步骤。此外，个别线粒体的可视化是唯一可能通过电子显微镜，它是不适合于线粒体功能的分析。话虽这么说，我们认识到，从组织线粒体的分离导致线粒体网络的破坏，这可能会影响线粒体功能的一些元素。然而，分离的线粒体的功能性方面，从组织中被类似地处理的比较仍然有效。 的组织来源isolat免疫标记的这个方法编线粒体和随后通过流式细胞仪分析可用于快速和量化的方法来检测和监控位于外膜的蛋白质的存在。检测高度丰富的线粒体蛋白（如Mfn2的）是可能的这种技术。同样，该方法能够检测低丰度的蛋白质，只附着在线粒体膜中的疾病，如肌萎缩侧索硬化症（ALS）11的上下文中的错误折叠的SOD1。此外，这种方法可能是有用的，以监视瞬时与线粒体表面相关联的，作为其正常功能的一部分的蛋白质。实例包括动力素相关蛋白-1（DRP1），即招募到线粒体，促进线粒体裂变12和肿瘤坏死因子受体相关因子6（TRAF6），其易位至线粒体，以增加线粒体活性氧物种的胞质蛋白的先天免疫应答13的一部分。 目前该技术是适合仅对位于线粒体表面蛋白，如标准透协议内标签需要的洗涤剂的破坏线粒体的结构完整性。虽然有许多可能的试剂已经尝试（未发表），该免疫标记协议的进一步优化，仍然需要使这个协议适合于检测内线粒体成分。 该技术具有广泛的应用，并且可以被用于检测在存在或招募的一种或多种蛋白以根据不同的实验范式线粒体。此外，线粒体可以共同标记有两种不同的抗体，以及具有荧光指示器11。其它荧光探针也可以掺入到表征线粒体功能的其它方面。例如，可商购的染料并监控线粒体pH为14 </s向上&gt;，钙的摄取与钙绿-5N 15，和ATP水平16顷可能的。

线粒体是经过多轮的裂变和聚变，被运到高能源需求的网站，并迅速生理刺激反应1高动态性的细胞器。因为人们越来越认识到不同组织内的线粒体，甚至不同的细胞区室，具有不同的功能配置文件，需要新的方法来识别这些不同的线粒体亚群。 显微术提供了一种方法，通过个别线粒体可以可视化和蛋白质在或在线粒体可以通过免疫2来确定是否存在。然而，用这种方法定量分析是劳动密集和更适合用永生化或原代细胞系的实验。来源于组织的个体的线粒体的研究是显著更加困难和最方法不允许以易于识别线粒体亚群与evalu并发通报BULLETIN线粒体功能3。 为了解决这一难题，一种新颖的方法来immunolabel线粒体分离从啮齿动物组织中，并随后分析通过流式细胞术已经被开发出来。这允许快速检测并定位于线粒体外膜，其相比于分析由显微镜蛋白质的定量，少得多的劳动密集的，并允许以十万计的线粒体中的单个样品的分析。此测定法可用于监测被认为是组成性存在于线粒体的线粒体外膜蛋白质的命运和相对量，蛋白质的募集到线粒体表面，或错误定位在病理条件下，线粒体蛋白的检测。此外，以往的荧光指示剂染料的掺入允许的线粒体功能的某些方面，在不同的线粒体同时评价亚群。

<table border="0" cellpadding="0" cellspacing="0" width="1276">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:349px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:543px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Rats</td>
			<td style="width:349px;">Charles River</td>
			<td style="width:119px;">Strain code 400</td>
			<td style="width:543px;">Adult (9 weeks to 18 weeks) male or female rats can be used for the isolation protocol. Weight of rats is dependent on gender and age (males between 300 to 500 g and females between 200 to 350 g) are typically used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dounce homogenizer</td>
			<td style="width:349px;">Kontes Glass Co.&#160;</td>
			<td style="width:119px;">885450-0022</td>
			<td>Duall 22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Microcentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall Legend Micro 17 R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Ulara-Clear Ultracentrifuge tubes</td>
			<td style="width:349px;">Beckman Coultier</td>
			<td style="width:119px;">344057</td>
			<td>Transparent, thin walled&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sorvall Ultracentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall WX UltraSeries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">AH-650 rotor and buckets&#160;&#160;</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">Opti-prep</td>
			<td style="width:349px;">Axis-Shield</td>
			<td style="width:119px;">1114542</td>
			<td>Iodixanol, density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Fatty acid free Bovine Serum Albumin</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8806</td>
			<td>Must be fatty acid free for mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sodium succinate dibasic hexahydrate&#160;&#160;</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">S9637</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:267px;">Rabbit anti-Mitofusin2 antibody</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">M6319</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:267px;">Rabbit IgG</td>
			<td style="width:349px;">Jackson Immuno Research&#160;</td>
			<td style="width:119px;">011-000-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Anti-Rabbit IgG PE</td>
			<td style="width:349px;">eBioscience</td>
			<td style="width:119px;">12-4739-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Micro titer tube</td>
			<td style="width:349px;">Bio-Rad</td>
			<td style="width:119px;">223-9391</td>
			<td>For sample acquisition by flow cytometry&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoTrackerGreen (MTG)</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M7514</td>
			<td>100 nM: Ex490 nm/Em516 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">TMRM</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">T668</td>
			<td>100 nM: Ex548 nm/Em574 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">CCCP</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">C2759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoSOX Red</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M36008</td>
			<td>5 &#181;M: Ex540 nm/Em600 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Antimycin A</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8874</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">LSR II flow cytometer</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BS FACSDiva Software</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FlowJo</td>
			<td style="width:349px;">TreeStar Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td>Software used for analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">BCA protein assay kit&#160;&#160;</td>
			<td style="width:349px;">Pierce/Thermo Scientific</td>
			<td style="width:119px;">23225</td>
			<td style="width:543px;">Bradford assay is not recomended as it is not compatible with high concentrations of SDS</td>
		</tr>
	</tbody>
</table>

immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria

方法来检测和监测线粒体外膜蛋白成分在动物组织是研究线粒体生理和病理生理至关重要。该协议描述了线粒体啮齿动物组织中分离出免疫标记是通过流式细胞仪检测的技术。线粒体是从啮齿动物的脊髓中分离并进行快速浓缩步骤，以除去髓磷脂，来自神经组织制备的线粒体级分中的主要污染物。分离的线粒体，然后标记所选择的抗体和荧光标记的二抗。分析流量通过与线粒体特异性染料，其次是检测和免疫标记蛋白定量染色流式细胞仪验证线粒体制剂的相对纯度。该技术具有快速，量化和高吞吐量，允许成千上万的每个样本的线粒体的分析。它可适用于评估新颖p在正常生理条件下，在线粒体表面roteins以及病理过程中，可能会成为错误定位到该细胞器的蛋白质。重要的是，这种方法可以被耦合到荧光指示剂染料对线粒体亚群的某些活动的报告，并为从中枢神经系统（脑和脊髓）以及肝线粒体可行的。

从大鼠脊髓源性线粒体可以免疫标记与靶向Mitofusin2（Mfn2的），牵连线粒体8的外膜的融合蛋白的抗体。以下隔离和标签带Mfn2的特异性抗体和荧光标记的二抗，线粒体通过流式细胞术（ 图1）进行处理。以下的数据采集，样品是使用流式细胞分析软件，由第一可视化上的点图（ 图2A）的所有收集的事件进行分析。当事件被绘制在FSC，宽度（W）相对于面积（A）（ 图2B）的双峰和单峰是有区别的。一旦汗衫被选择时，栅极线粒体人口经由FSC / SSC（ 图2C），并验证事件由染色阳性线粒体特异性染料的数量共绘制未染色样品的直方图用沾有线粒体SPE样品cific染料。以确定的线粒体事件，放置栅极处的两个峰（ 图2D）的交叉点。对于脊髓制剂，通常&gt; 90％的事件都积极为线粒体特异染料。对于其他组织如肝，〜98％的事件将与染料标记（数据未示出）。仅选择该标签正对线粒体特异性染料的事件后，通过选择一个栅极（Mfn2的+），其包括1％或更少的同种型的标记（ 图2E， 左 ）确定与同种型对照背景标记。均匀地施加该栅极到标记抗体Mfn2的确定线粒体与Mfn2的比例存在于线粒体外膜（ 图2E， 右 ）的所有样本。在该实验中，线粒体的30％来自于脊髓标签的正面为Mfn2的。这里要注意的是，尽管Mfn2的被认为是一种广泛表达的MIT是很重要ochondrial外膜蛋白，其先前尚未定量。此外，Mfn2的在培养的细胞的免疫细胞化学分析，显示个别线粒体9的非均质标记。它也有可能是在Mfn2的表位是不可用的，由于翻译后修饰或由于与自身相互作用或其它结合配偶。值得注意的是，使用的合成肽的N-末端（氨基酸38-55）中的生成在本研究的抗体。有Mfn2的的预测剪接变体缺乏的第一302个氨基酸，尽管该变体还没有被实验确认（的UniProt数据库）。因此，该测定法无法检测可变剪接Mfn2的缺乏N-末端序列，给定所使用的抗体。 线粒体跨膜电位（ΔΨ 米 ）和超氧化物的产生可以在该测定中进行评估。穿过线粒体内膜，有负责WH分离ICH驱动器通过氧化磷酸化生成ATP。 TMRM是阳离子染料，其在线粒体内的膜电位依赖性5累加的，因此可以用作线粒体跨膜电位的记者。大多数线粒体（95％）的是TMRM阳性染色后，相比于未染色的对照（ 图3A）。然而，当解偶联剂CCCP添加有在线粒体能够保留TMRM（ 图3A）的数量显著减少。 CCCP允许离子自由通过跨线粒体膜，实质上破坏电荷的分离和去极化膜。 线粒体释放过氧化物作为氧化磷酸化，从复合物I和电子传递链的三正常副产品。线粒体超氧可以通过被靶向线粒体，成为荧光follo膜渗透性染料进行测量翼超7的反应。功能性线粒体产生超氧相比未染色的样品（ 图3B）基底量。此外，复合物III抑制剂抗霉素A的产量增加超，所看到的右移在花期和一些线粒体较高（通常约10％）是荧光与此线粒体超氧指示剂染料（ 图3B）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51887/51887fig1highres.jpg" width="500" /> 图1原理图的隔离，免疫标记和线粒体的分析。步骤1:收集组织，均质步骤2:在分离过程包含八个离心步骤。髓鞘，中枢神经系统组织的主要遏制是由碘克沙醇（密度梯度介质）和CENTRI稀释线粒体除去fuging，导致髓鞘浮到该管的顶部，而线粒体沉淀步骤3:在分离和定量，线粒体被堵塞，标记的第一抗体和洗涤。缀合至荧光基团的二次抗体，然后加入。未结合的抗体洗去步骤4:此时的荧光染料对线粒体的纯度，或线粒体功能的报告可以添加步骤5:线粒体是现在可以通过流式细胞仪检测。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51887/51887fig2highres.jpg" width="500" /> 图2。策略分离线粒体通过流动分析仪。前向散射（FSC）和侧向散射（SSC）的电压必须为小型活动进行调整，用对数模式这两个参数。 FSC宽度（FSC-W）的数据必须是收集排除双峰。请注意，在大多数流式细胞仪，默认设置为FSC和SSC是线性模式（A），可视化所有收集的事件上点图（B）双峰，2线粒体通过激光在同一时间的推移，可以区分汗衫绘制FSC与FSC-W（线性模式）。事件被排除在外，如果FSC-W的值是在大多数的事件， 即，那些致密云的一部分的两倍以上的平均FSC-W的值。在这道门槛的事件门为单峰（C）同样，在可视化的点积的事件，并在余下的赛事门（D）画出未染色样品的直方图（固体，灰色，填充）和样品沾有线粒体特定的染料（MSD:固体，绿色）。门染色阳性的MSD（MSD +），（五）同型对照，兔IgG的直方图的事件（虚线，黑色）和Mfn2的标记的样品（固体，粉红色）。将门以产生≤1％Mfn2的+对同型对照峰值，这同一门适用于实验样本，以确定事件中Mfn2（Mfn2的+）标记阳性的百分比。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51887/51887fig2highres.jpg" target="_blank">请点击这里查看这个放大版图。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51887/51887fig3highres.jpg" width="500" /> 图3含量测定线粒体跨膜电位（ΔΨ 米）和超氧化物的产生由流动分离线粒体术。 （一）在基础条件的所有活动将线粒体与TMRM（固体，黑色）的标签，相比于未染色的对照（固体，灰色，填充），因为染料聚集在线粒体跨膜电位。阿迪<html>该protonophore CCCP（虚线，蓝）的灰消散跨膜电位使线粒体去极化和保留较少的染料相比，在基础条件。数据报告为增量平均荧光强度（ΔMFI），这是未染色的对照，从该样品的平均荧光强度减去的平均荧光强度（B）在基础条件下，线粒体产生超氧化物如氧化的副产物磷酸化。超的这个线粒体源可检测与线粒体超氧化物指标，（MitoO 2:固体，黑色）相比，未染色的对照（固体，灰色，填充）。此外，复合物III抑制剂，抗霉素A的（虚线，橙色）结果增加超氧化物生产，与基线水平相比。数据报告为染色阳性细胞为MitoO 2指标的百分比。K"&gt;请点击这里查看该图的放大版本。

Watch this Scientific Journal Video about Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria at JoVE.com

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

这里描述的是这样一种方法来检测并通过免疫标记线粒体分离从啮齿动物的组织和通过流式细胞仪分析的量化线粒体外膜蛋白质。该方法可以被扩展以评估线粒体亚群的功能方面。

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

immunodetection-outer-membrane-proteins-flow-cytometry-isolated

Nanyang Technological University

Research

JoVE Journal

Biology

15.4K 次观看.  Université de Montréal, CRCHUM. 这里描述的是这样一种方法来检测并通过免疫标记线粒体分离从啮齿动物的组织和通过流式细胞仪分析的量化线粒体外膜蛋白质。该方法可以被扩展以评估线粒体亚群的功能方面。 

视频: Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

科研

生物学

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

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 ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.