Name: measuring mitochondrial substrate flux in recombinant perfringolysin o-permeabilized cells
Uploaded: 2021-08-13T14:30:00
Description: Watch this Scientific Journal Video about Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells at JoVE.com

作者感谢Hradec Králové医学院生理学系和第三医学院病理生理学系的工作人员在化学品和样品制备方面的帮助。这项工作得到了查理大学拨款计划PROGRES Q40/02，捷克卫生部拨款NU21-01-00259，捷克科学基金会拨款18-10144和INOMED项目CZ.02.1.01/0.0/0.0/18_069/0010046的支持，该项目由捷克共和国教育，青年和体育部和欧盟资助。

1. 化验前一天
<ol><li>试剂和底物的制备。<ol>
<li>线粒体测定溶液（MAS）：按 表1所述制备所有试剂的储备溶液。将甘露醇和蔗糖的储备加热至37°C以完全溶解。混合试剂以制备2x MAS，然后将混合物加热至37°C。 用5N KOH调节pH至7.4（〜7 mL），然后加水使体积达到1 L.过滤灭菌并将等分试样储存在-20°C直至测量日。</li>
		<li>牛血清白蛋白（5%BSA）：将5克BSA溶解在磁力搅拌器上，加入90毫升?...

<ol>
	<li>Gerencser, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+microplate-based+respirometry+with+correction+for+oxygen+diffusion.">Quantitative microplate-based respirometry with correction for oxygen diffusion.</a> Analytical Chemistry. 81 (16), 6868-6878 (2009).</li><li>Murphy, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function,+biology,+and+role+in+disease:+A+scientific+statement+from+the+American+Heart+Association.">Mitochondrial function, biology, and role in disease: A scientific statement from the American Heart Association.</a> Circulation Research. 118 (12), 1960-1991 (2016).</li><li>Owen, O. E., Kalhan, S. C., Hanson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+key+role+of+anaplerosis+and+cataplerosis+for+citric+acid+cycle+function.">The key role of anaplerosis and cataplerosis for citric acid cycle function.</a> Journal of Biological Chemistry. 277 (34), 30409-30412 (2002).</li><li>Nicholls, D. G., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioenergetics 3. , (2002).</li><li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>Sta&#328;kov&#225;, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+mitochondrial+substrate+flux+in+a+mouse+model+of+nonalcoholic+fatty+liver+disease.">Adaptation of mitochondrial substrate flux in a mouse model of nonalcoholic fatty liver disease.</a> International Journal of Molecular Sciences. 21 (3), 1101 (2020).</li><li>Salabei, J. K., Gibb, A. A., Hill, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+measurement+of+respiratory+activity+in+permeabilized+cells+using+extracellular+flux+analysis.">Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.</a> Nature Protocols. 9 (2), 421-438 (2014).</li><li>Divakaruni, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazolidinediones+are+acute,+specific+inhibitors+of+the+mitochondrial+pyruvate+carrier.">Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (14), 5422-5427 (2011).</li><li>Divakaruni, A. S., Rogers, G. W., Murphy, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+function+in+permeabilized+cells+using+the+seahorse+XF+analyzer+or+a+Clark-type+oxygen+electrode.">Measuring mitochondrial function in permeabilized cells using the seahorse XF analyzer or a Clark-type oxygen electrode.</a> Current Protocols in Toxicology. 60, 1-16 (2014).</li><li>Elkalaf, M., T&#367;ma, P., Weiszenstein, M., Pol&#225;k, J., Trnka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+probe+Methyltriphenylphosphonium+(TPMP)+inhibits+the+Krebs+cycle+enzyme+2-Oxoglutarate+dehydrogenase.">Mitochondrial probe Methyltriphenylphosphonium (TPMP) inhibits the Krebs cycle enzyme 2-Oxoglutarate dehydrogenase.</a> PLoS One. 11 (8), 0161413 (2016).</li><li>Rogers, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PLoS One. 6 (7), 21746 (2011).</li></ol>

该协议是对先前发表的研究7、8、9、10和产品用户指南的修改。与制造商的协议相比，使用2x MAS代替3x MAS，因为2× MAS更容易溶解，并且在冷冻后不会形成沉淀。冷冻的 2 倍 MAS 等分试样可储存长达 6 个月，并显示一致的结果。另一个区别是将ADP包含在2x MAS的组分中，并从公式中省略BSA。含有BSA的解决?...

基于微孔板的呼吸测定法通过能够研究小样本量1的细胞呼吸，彻底改变了线粒体研究。细胞呼吸通常被认为是线粒体功能或"功能障碍"的指标，尽管线粒体功能范围超出了能量产生2。在有氧条件下，线粒体通过分解并将这些底物转化为可以为柠檬酸循环3 提供燃料的代谢中间体来提取储存在不同底物中的能量（图1）。底物的连续通量对于柠檬酸循环的流动至关重要，以产生高能"电子供体"，其将电子传递到电子传递链，该传递链产生穿过线粒体内膜的质子梯度，使ATP合成酶能够将ADP磷酸化为ATP4。因此，测定线粒体呼吸的实验设计必须包括样品性质（完整细胞，透化细胞或分离的线粒体）和线粒体底物。
细胞储存着本土底物5，线粒体同时氧化几种类型的底物6，这使得对在完整细胞上进行实验的结果的解释变得复杂。研究线粒体氧化所选底物能力的常用方法是分离线粒体或透化所研究的细胞5。虽然分离的线粒体是定量研究的理想选择，但分离过程很费力。它面临着技术难题，例如需要大样本量，纯度产量和该技术的再现性5。透化细胞为线粒体分离的缺点提供了解决方案;然而，洗涤剂性质的常规通透性剂不是特异性的，并且可能损害线粒体膜5。
重组鼓气溶酶O（rPFO）作为选择性质膜透化剂7提供，并在7，8，9，10的几项研究中成功地与细胞外通量分析仪结合使用。我们使用rPFO修改了使用XFe96细胞外通量分析仪筛选线粒体底物通量的方案。在该协议中，比较了两种细胞表型中的四种不同的底物氧化途径，同时对每种测试材料具有足够的重复和适当的控制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adinosine 5&#8242; -diphosphate monopotassium salt dihydrate</td>
			<td>Merck</td>
			<td>A5285</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Merck</td>
			<td>A8674</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Merck</td>
			<td>A3803</td>
			<td>store at 2 - 8 &#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td>Merck</td>
			<td>C2920</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Merck</td>
			<td>D8418</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Merck</td>
			<td>63559</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Merck</td>
			<td>03777</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Merck</td>
			<td>H7523</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>L(-)Malic acid disodium salt</td>
			<td>Merck</td>
			<td>M9138</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>L-Glutamic acid sodium salt hydrate</td>
			<td>Merck</td>
			<td>G5889</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Magnissium chloride hexahydrate</td>
			<td>Merck</td>
			<td>M2670</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Merck</td>
			<td>O4876</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Palmitoyl-DL-carnitine chloride</td>
			<td>Merck</td>
			<td>P4509</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Merck</td>
			<td>484016</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Merck</td>
			<td>P5655</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Merck</td>
			<td>R8875</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Seahorse Wave Desktop Software</td>
			<td>Agilent technologies</td>
			<td></td>
			<td>Download from www.agilent.com</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent technologies</td>
			<td>102416-100</td>
			<td>XFe96 sensor cartridges and XF96 cell culture microplates</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Merck</td>
			<td>P2256</td>
			<td>store at 2 - 8 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium succinate dibasic hexahydrate</td>
			<td>Merck</td>
			<td>S2378</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck</td>
			<td>S7903</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Merck</td>
			<td>W3500</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>XF calibrant</td>
			<td>Agilent technologies</td>
			<td>100840-000</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>XF Plasma membrane permeabilizer</td>
			<td>Agilent technologies</td>
			<td>102504-100</td>
			<td>Recombinant perfringolysin O (rPFO) - Aliquot and store at -20 &#176;C</td>
		</tr>
	</tbody>
</table>

measuring mitochondrial substrate flux in recombinant perfringolysin o-permeabilized cells

线粒体底物通量是每种细胞类型的显着特征，其成分（如转运蛋白，通道或酶）的变化与几种疾病的发病机制有关。线粒体底物通量可以使用完整细胞、透化细胞或分离的线粒体进行研究。研究完整的细胞由于同时氧化不同的底物而遇到几个问题。此外，几种细胞类型包含不同底物的内部存储，使结果解释复杂化。线粒体分离或使用透化剂等方法不易重复。从小样品中分离出具有完整膜的纯线粒体是有问题的。使用非选择性透化剂会导致不同程度的不可避免的线粒体膜损伤。重组透气溶酶O（rPFO）作为更合适的透气剂提供，因为它能够选择性地透化质膜而不影响线粒体完整性。当与微孔板呼吸法结合使用时，它允许在一个实验中使用最少数量的细胞，在一个实验中用足够的重复来测试几种线粒体底物的通量。在这项工作中，该协议描述了一种比较两种不同细胞表型或基因型的线粒体底物通量的方法，并且可以定制以测试各种线粒体底物或抑制剂。

首先将结果归一化为基线呼吸的第二次测量值，以将值显示为耗氧率百分比 （OCR%）。测定结果如图5、图6、图7和图8所示。必须为每个组分配适当的背景孔，并停用其他组的背景孔。图5显示，治疗组具有较高的琥珀酸盐诱导呼吸率。A549细胞对二甲双胍治疗的?...

Watch this Scientific Journal Video about Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells at JoVE.com

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

在这项工作中，我们描述了一种改进的方案，以使用重组产气溶酶O与基于微孔板的呼吸测定法一起测试线粒体呼吸底物通量。通过该协议，我们展示了二甲双胍如何影响两种不同肿瘤细胞系的线粒体呼吸。

测量重组透气溶性细胞中线粒体底物通量 O-透化细胞

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or ...

因为它测试线粒体底物通量，这些通量可能会受到不同病理或治疗的影响。例如，今天我们将用它来揭示癌细胞对二甲双胍治疗的反应。它需要最少数量的细胞，同时对每种不同的材料进行足够的重复和适当的控制。该分析仪仅用于研究用途。该技术可以识别线粒体代谢中的错误，并可用于评估影响线粒体的战士药物。演示该程序的将是Karolina Vaneckova，她是我实验室的本科生和研究技术人员。以每孔20， 000个细胞的密度开始接种A549细胞，在色谱柱中加入2至11个海马XF 96细胞培养微孔板。将第 1 列和第 12 列留空为背景井。用等体积的细胞培养基填充空白孔，然后将细胞在37摄氏度下在加湿的培养箱中孵育，其中含有5%二氧化碳用于细胞附着。三到四小时后，在孔中加入100微升细胞培养基。用一毫摩尔二甲双胍处理实验组第7至11柱，用等体积的无菌蒸馏水处理对照组，然后将板返回培养箱。为了给传感器补水，将每口井200微升无菌水移入公用设施板中。然后小心地将传感器盒放回，同时将传感器浸入水中。将墨盒在37摄氏度的无二氧化碳培养箱中孵育至第二天。打开分析仪和控制单元。启动仪器控制和数据采集软件，并按照文本手稿中所述设计测定方案。在组定义下，创建四个注入策略，其中端口 A 根据注入的底物而不同，并在底物或缩写之后命名策略。将寡霉素分配到端口B，FCCP分配到端口C，将鱼藤酮抗霉素A混合物分配到端口D.创建并命名八个组，在板图下，将组分配给相应的孔。然后将协议另存为即用型模板。保持分析仪开关打开，使温度在一夜之间稳定下来。第二天，丢弃公用设施板中的水，并在公用设施板中每孔添加200微升预热的校准剂。将墨盒返回到无二氧化碳培养箱，直到测定时间。保持培养箱内的湿度源，关闭或将风扇速度降至最低，以避免校准剂快速蒸发。用预热的两次线粒体测定溶液，5%BSA和无菌水制备5毫升底物和抑制剂的工作溶液，如文本手稿中所述。接下来，按照税务手稿中所述，在注射器端口中加载底物和抑制剂。要在运行检测选项卡下进行校准，请单击开始运行以开始检测。插入加载的审查盒并等待校准完成。通过将两倍线粒体测定溶液，无菌水和5%BSA混合在50毫升管中，制备20毫升测定培养基。加入两微升10微摩尔重组产气溶素O，以达到1纳摩尔的浓度。并通过轻柔移液重新悬浮混合物，避免摇晃和涡旋。将管子在37摄氏度下孵育直至使用。使用多通道移液器洗涤细胞和空空白孔两次，使用预热的无钙和镁PBS溶液。丢弃PBS并加入180微升预热的测定培养基以进行细胞透化。通透后，立即将校准传感器盒的实用程序板替换为含有透化细胞的细胞板并开始测量。治疗组琥珀酸盐诱导呼吸的发生率较高。A549细胞对二甲双胍治疗的反应高于Hep G2。对于丙酮酸苹果酸盐诱导的呼吸，与Hep G2细胞相比，A549细胞在5至15分钟之间显示出增加的诱导作用。谷氨酸苹果酸盐诱导呼吸和棕榈酰肉碱苹果酸盐诱导呼吸的结果相似。达到合适的底物和抑制剂浓度。必须将正确的卷加载到正确的端口中。当确定某种底物的代谢途径发生变化时，有必要评估参与该途径的酶和转运蛋白的功能。

Research

JoVE Journal

Biology

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

科研

生物学

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Measuring Glutathione-induced Feeding Response in Hydra

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

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

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...