Name: using real-time cell metabolic flux analyzer to monitor osteoblast bioenergetics
Uploaded: 2022-03-01T12:45:00
Description: Watch this Scientific Journal Video about Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics at JoVE.com

这项工作得到了美国国立卫生研究院（NIH）国家关节炎和肌肉骨骼和皮肤病研究所（NIAMS）拨款AR072123和国家衰老研究所（NIA）拨款AG069795（ERR）的支持。

所有程序均基于范德比尔特大学医学中心机构动物护理和使用委员会的指导方针和批准。
1. 试剂制备和测定设置
<ol><li>骨髓基质细胞的分离和培养（另见上一篇文章30）。<ol>
<li>制备完全α最小必需培养基（αMEM）细胞培养基，方法是用10%FBS（胎牛血清），100 U / mL青霉素和100μg/ mL链霉素补充具有α修饰的最小必需培养基。</li>
		<li>通过修剪0.6 mL...

<ol>
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实时细胞代谢通量分析仪可用于探索不同条件下的细胞能量学。该协议说明了BMSC的有效分离，在适当的细胞培养板中培养细胞，以及它们分化为成熟的成骨细胞，这可用于使用细胞外通量分析仪进行各种测定。此外，还详细解释了实时细胞代谢通量测定的关键步骤，包括传感器盒的水合，注射端口的加载，执行测定，数据归一化和数据分析。该测定评估成骨细胞对不同线粒体和糖酵解抑制剂的反...

成骨细胞形成的骨骼伴随着破骨细胞对骨骼的协调破坏或再吸收。成骨细胞骨形成和破骨细胞再吸收之间的平衡是描述骨转换或重塑的耦合过程，这对于骨骼稳态至关重要。成骨细胞功能障碍导致骨骼形成受损并导致各种疾病，包括骨质疏松症1，2，3。骨髓基质干细胞（BMSCs）到成骨细胞前体和成熟成骨细胞的 离体/体外 分化导致矿化骨基质随着时间的推移在培养血管中形成和沉积4，5，6。成骨细胞形成的这种骨骼需要大量的细胞能量。具体而言，胶原蛋白的合成和分泌已被证明严重依赖于细胞ATP：ADP比率，并且可能，矿化囊泡运输和分泌需要额外的ATP7，8，9，10，11。许多研究人员已经证明，成骨细胞生成和成骨细胞功能的过程需要足够的能量供应来满足骨形成的代谢需求12，13，14，15，16。因此，该方法的目标是使用实时细胞代谢通量分析仪表征原代，小鼠基质细胞在整个成骨细胞分化过程中的生物能量状态。这些技术有助于更好地了解骨骼稳态，这可能最终导致能够改善骨骼疾病的新型治疗方案的发展。
实时细胞代谢通量分析仪可用于测量活成骨细胞的耗氧速率（OCR）和细胞外酸化速率（ECAR），分别对应于线粒体和糖酵解ATP的产生。这种方法的基础是，在糖酵解过程中，每个乳酸释放一个H + 离子，将葡萄糖转化为乳酸盐，这会改变ECAR值中反映的培养基pH值。相反，在TCA（三羧酸）循环期间， 通过 线粒体的氧化磷酸化通过利用或消耗氧气产生CO2 ，因此监测OCR反映了这种代谢过程。该分析仪同时实时测量细胞外微环境中的OCR和ECAR，这在研究细胞生物能量学时具有巨大的潜力6，17。此外，进行这些测定相对简单，并且易于根据实验目标进行定制。已经采用类似的技术来进一步了解免疫系统的T细胞代谢调节18，19，癌症的发生和进展20，以及导致代谢综合征的多种其他细胞类型21，22。
与替代技术相比，实时代谢通量分析仪的优势包括：（1）能够实时测量活细胞的细胞生物能量，（2）能够对相对较少的细胞进行测定（需要低至5，000个细胞），（3）注射端口在高通量96孔系统中并行操作多个处理，（4）使用无放射性标记的自动细胞成像仪进行归一化18，23，24.以下方法旨在提供使用分析仪在整个成骨细胞分化过程中监测小鼠BMSC中细胞生物能量学的广义但详细的描述。它将包括常规进行的测定;然而，与许多技术和方法一样，强烈建议各个实验室确定其实验的具体细节。
可选择测定和不同类型的测定：提供多种测定试剂盒和试剂，用于研究细胞的生物能量学，同时确保实验结果的可靠性和一致性。此外，桌面软件还提供可轻松定制的分析模板。该测定可以根据用户测量不同代谢参数的需要来定义。这些测定可以根据实验目标和/或科学问题以各种方式进行修改。例如，使用四个进样端口，可以将多种化合物注射到测定培养基中，以分析特定于每个代谢途径的细胞反应。
细胞能量表型测试： 该测定法测量活细胞的代谢表型和代谢潜力。该测定也被推荐作为获得通路特异性代谢的广义概念的第一步。注入寡霉素A-ATP合酶抑制剂和羰基氰化物4-（三氟甲氧基）苯腙（FCCP）-线粒体解偶联剂的混合物以了解细胞能量电位。注射寡霉素A抑制ATP的合成，导致糖酵解速率（ECAR）的增加，使细胞能够满足其能量需求;另一方面，由于线粒体膜的去极化，FCCP的注射导致更高的OCR。从本质上讲，该测定描绘了基础代谢呼吸，并在双重注射，推或应激之后，代谢反应。基于这些参数，软件然后通过将细胞分类为有氧，静止，糖酵解或能量状态来绘制细胞的OCR和ECAR，时间跨度25，26。
ATP实时产率测定： 这测量细胞ATP同时从糖酵解和线粒体呼吸产生的。该测定定量测量来自两个能量途径的代谢变化，并提供有关线粒体和糖酵解ATP产生速率随时间变化的数据。该测定获得基础OCR和ECAR数据，然后通过注射寡霉素A计算线粒体ATP产生速率，通过注射鱼藤酮+抗霉素A混合物计算糖酵解ATP产生速率（完全抑制线粒体功能），导致线粒体酸化17，27。
细胞线粒体负荷试验（或细胞丝粒体负荷试验）： 这通过ATP连接的呼吸测量线粒体功能，量化细胞生物能量，识别线粒体功能障碍，并测量细胞对压力的反应。一次测定即可获得各种参数，包括基础和备用呼吸能力、ATP 相关呼吸、最大呼吸和非线粒体耗氧量。该测定涉及序贯注射寡霉素A，FCCP（线粒体解偶联剂），鱼藤酮/抗霉素A抑制剂的混合物，以有效分析这些抑制剂对线粒体功能的影响28。
柔韧性水母燃料柔性测试： 这通过三种主要线粒体燃料的存在和不存在抑制剂的氧化来测量线粒体呼吸速率。葡萄糖，谷氨酰胺和脂肪酸的顺序抑制有助于测量细胞的依赖性，容量和柔韧性以及细胞在各种细胞途径中的依赖性，以满足能量需求。当线粒体不能通过氧化其他燃料来满足阻塞的靶向途径的需求时，细胞进入依赖状态。细胞的容量是通过抑制其他两种替代途径，然后抑制感兴趣的途径来计算的。细胞的灵活性有助于了解线粒体补偿和满足抑制途径的燃料需求的能力。它是通过从单元的容量中减去单元的依赖性来计算的。独立使用三种不同的抑制剂或作为两种抑制剂的混合物来有效计算测定参数。2-氰基-3-（1-苯基-1H-吲哚-3-基）-2-丙烯酸（UK5099）通过阻断糖酵解中的丙酮酸盐载体来抑制葡萄糖的氧化。双-2-（5-苯基乙酰氨基-1，3，4-噻二唑-2-基）（BPTES）乙基硫醚抑制谷氨酰胺氧化途径，依托莫西抑制长链脂肪酸的氧化29。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63142/63142fig01.jpg"> 图1：用于培养和制备成骨细胞进行分析的方法示意图。 从长骨中分离出小鼠BMSC，培养并接种在96孔板中，密度为25，000个细胞/ 孔。当这些细胞达到80%-100%汇合以开始分化时，就开始在成骨细胞特异性培养基中培养这些细胞。测定在不同的分化阶段进行。在测定前一天将筒板水合。在测定当天，根据测定要求将不同的抑制剂注射到传感器盒的端口中，并将校准缓冲液添加到96孔校准板中。校准后，进行实时细胞代谢通量测定，然后使用微孔板成像仪对细胞培养微孔板进行成像，以使用细胞计数对实时细胞代谢通量分析仪数据进行归一化。 <a href="https://www.jove.com/files/ftp_upload/63142/63142fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-propenoic acid</td>
			<td>Sigma - Aldrich</td>
			<td>PZ0160</td>
			<td>UK5099</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma - Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide</td>
			<td>Sigma - Aldrich</td>
			<td>SML0601</td>
			<td>BPTES</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td>Sigma - Aldrich</td>
			<td>C2920</td>
			<td>FCCP</td>
		</tr>
		<tr>
			<td>Cytation 5 imaging reader</td>
			<td>BioTek</td>
			<td>N/A</td>
			<td>Microplate imager</td>
		</tr>
		<tr>
			<td>Etomoxir sodium salt hydrate</td>
			<td>Sigma - Aldrich</td>
			<td>E1905</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 Solution (20 mM)</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma - Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic Acid-Albumin from bovine serum</td>
			<td>Sigma - Aldrich</td>
			<td>O3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin A - 5 mg</td>
			<td>Sigma - Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma - Aldrich</td>
			<td>R8875-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 1.0 M Glucose Solution</td>
			<td>Agilent Technologies</td>
			<td>103577-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 100mM Pyruvate Solution</td>
			<td>Agilent Technologies</td>
			<td>103578-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 200mM Glutamine solution</td>
			<td>Agilent Technologies</td>
			<td>103579-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF DMEM media</td>
			<td>Agilent Technologies</td>
			<td>103575-100</td>
			<td>DMEM assay media eith 5mM HEPES, pH 7.4, without phenol red, sodium bicarbonate, glucose, pyruvate, and L-glutamine</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent Technologies</td>
			<td>S7800B</td>
			<td>Real- Time Metabolic flux analyzer</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent Technologies</td>
			<td>102416-100</td>
			<td>Includes XFe96 Sensor cartridges, Cell culture microplates, and Seahorse XF Calibrant solution</td>
		</tr>
		<tr>
			<td>The Cell imaging 1.1.0.11 software</td>
			<td>Agilent Technologies - BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wave software 2.6.1</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerol phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422-50G</td>
			<td></td>
		</tr>
	</tbody>
</table>

using real-time cell metabolic flux analyzer to monitor osteoblast bioenergetics

成骨细胞的骨形成是正确获得骨骼和骨更新以维持骨骼稳态并最终防止骨折的必要过程。为了优化峰值骨量和对抗各种肌肉骨骼疾病（即绝经后骨质疏松症，神经性厌食症，1型和2型糖尿病），在骨生物学领域做出了令人难以置信的努力，以在整个分化过程中充分表征成骨细胞。鉴于成熟成骨细胞分泌基质蛋白和矿化囊泡的主要作用，已经注意到这些过程需要令人难以置信的细胞能量，或三磷酸腺苷（ATP）。整体细胞能量状态通常被称为细胞生物能量学，它包括一系列代谢反应，这些反应感知底物的可用性，以获得ATP以满足细胞需求。因此，目前的方法详细介绍了分离原代、小鼠骨髓基质细胞（BMSCs）的过程，并使用实时细胞代谢通量分析仪在成骨细胞分化的各个阶段监测其生物能量状态。重要的是，这些数据表明，在整个成骨细胞分化过程中，代谢谱发生了巨大变化。因此，使用这种生理相关的细胞类型需要充分了解细胞的生物能量状态如何调节整体功能。

<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63142/63142fig06.jpg"> 图6：常规检测的代表性图表，以了解对照组与治疗组的细胞生物能量谱及其各自的标准误差。该图表示对照组的糖酵解 （ECAR） 与线粒体呼吸 （OCR） 与两个治疗组 （n = 3）。注射寡霉素A和FCCP应激源可提高基线活性，由开放符号表示，而封闭符号?...

Watch this Scientific Journal Video about Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics at JoVE.com

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

实时细胞代谢通量测定使用pH和氧传感器测量耗氧速率和细胞外酸化速率，这对应于线粒体和糖酵解三磷酸腺苷的产生。该手稿解释了一种了解成骨细胞能量状态的方法以及细胞生物能量状态的表征和解释。

使用实时细胞代谢通量分析仪监测成骨细胞生物能量学

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

这种方法可用于确定细胞能量学，如ATP产生速率，以及实时确定细胞对特定代谢物的偏好。首先，加入200微升XF校准剂，并在测定前将实用板孵育至少一小时。使用补充的XF DMEM制备80毫升XF测定培养基。在冰上解冻寡霉素A，鱼藤酮和抗霉素A。上下移液化合物以在使用前溶解它们。向每个试管中加入三毫升制备的测定培养基，标记A和B.将26.4微升2.5毫摩尔寡霉素A加入管A和3.1微升12.67毫摩尔鱼藤酮，4.1微升9.4毫摩尔抗霉素A和30微升Hook污渍到管B中，在相应的注射口中加载10倍浓度的这些抑制剂。最后，20微升这些化合物将在200微升的测定培养基中加载到细胞中。从37摄氏度培养箱中取出细胞培养微孔板，并在显微镜下观察细胞。从水浴中取出测定培养基。用200微升预热的测定培养基轻轻洗涤细胞两次，每孔加入200微升测定培养基。在显微镜下检查细胞，以确保细胞保持粘附在孔上。确保D5和E8孔中的细胞粘附在一致的单层上，并且在洗涤步骤中没有被冲走。细胞成像软件使用这两个孔来设置自动对焦和自动曝光。在设备旁边的计算机中打开桌面软件。检查控制器软件左下角的连接状态。转到模板并选择XF ATP速率测定模板或适当的测定模板。选择屏幕顶部的组定义并定义组。选择板图布局并根据定义的组分配孔。验证仪器协议。确保添加的化合物正确列出，并包括项目信息以供将来参考。单击运行分析。这将提示选择结果文件存储位置。选择保存结果文件的位置。保存带有检测日期的文件，然后单击开始运行。将传感器盒和实用程序板放在托盘上，然后单击“我已准备好”以启动校准。在计算机上打开细胞成像软件。确保微孔板成像仪已打开，并且端口已连接到计算机。检查屏幕左下角的状态栏，确保温度设置为 37 摄氏度，并且连接状态应以绿色突出显示为就绪。扫描板条形码以启动成像过程。为细胞板提供一个名称，然后点击保存。明场和荧光图像将保存在此处。单击“执行明场扫描”，在下一个屏幕中，“板”和“扫描”菜单将显示成像选项。在检测之前，选择开始明场扫描。将细胞培养微孔板和板盖放在托盘支架上，并将A1孔与A1标记对齐。单击关闭托盘。明场图像采集出现在下一个带有板块图的屏幕中。单击“扫描所有孔”并扫描30至35分钟。校准完成后，软件将显示“称重传感器板”对话框。然后，单击“打开托盘”以用细胞培养微孔板替换实用程序托盘。请注意，细胞培养板位于微孔板成像仪中。扫描后，取出细胞培养微孔板并将其置于分析仪中以进行测定。然后，单击称重传感器板以启动测定。等到测定开始并显示估计的完成时间。分析完成后，软件将显示“卸载传感器盒”和“细胞板”对话框。单击“弹出”并从分析仪中取出细胞培养微孔板。小心地卸下传感器盒并盖上电池板盖。将出现“检测完成”对话框。然后，单击“查看结果”以打开测定结果文件或对数据进行归一化。测定完成后，用手持式条形码阅读器扫描板条形码。如果板材已经过成像，则不需要新名称。选择荧光和细胞计数。将细胞板放在托盘支架上，然后单击关闭托盘。在图像采集窗口中，选择扫描所有孔以开始成像。通过随机单击几个孔来查看成像和细胞成像应用程序中的荧光图像和细胞计数。荧光成像完成后，导出图像以供参考。成像和细胞计数完成后，打开结果文件，然后单击“归一化”。单击“导入”，然后选择“应用”，以便使用细胞计数自动对测定进行归一化。寡霉素A和FCCP应激源混合注射增加了对照组的基线活性。应激源在对照组和治疗组显示出显着高的能量水平。另一方面，治疗二具有相对较低的基线活性，并且细胞变得更加有氧。该图表明对照和实验细胞都使用糖酵解和线粒体氧化磷酸化来产生ATP。实验组显示通过糖酵解减少ATP生成，线粒体呼吸增加。治疗组基础呼吸率相对低于对照组。两组的呼吸和ATP产生率均降低，伴随着质子泄漏，随后注射寡霉素A。注射FCCP后，细胞的呼吸速率再次上升到其最大呼吸。最后注射鱼藤酮和抗霉素A再次降低OCR。第三次注射后，鱼藤酮和抗霉素A联合关闭线粒体呼吸，与对照组相比，治疗组的基础和最大呼吸相对没有变化。该图显示，对照组高度依赖葡萄糖途径，而谷氨酰胺氧化在对照组中是有效的。脂肪酸途径表明，治疗增加了细胞的整体容量。酸板上多个注射端口的可用性有助于我们确定多种药物在单个时间点对细胞能量的影响。

Research

JoVE Journal

Biology

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

科研

生物学

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

排序<em&gt;链霉菌</em&gt;细胞沉淀用一个复杂的对象参数分析仪和分选机

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

膜蛋白的实时测量：受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

沉默<em&gt; BRCA2</em&gt;以确定新的BRCA2调控的生物学功能在人体细胞培养

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...