Name: assessment of cellular bioenergetics in mouse hematopoietic stem and primitive progenitor cells using the extracellular flux analyzer
Uploaded: 2021-09-24T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer at JoVE.com

伦巴第实验室的工作得到了NIH（NIGMS R01GM101171，NIEHS R21ES032305），DoD（CA190267，CA170628，NF170044和ME200030）和Glenn医学研究基金会的支持。Li实验室的工作由NIH（NHLBI 5R01HL150707）支持。

所有脊椎动物实验均由密歇根大学动物使用和护理委员会批准并按照其进行。
1.测定前一天（总时间：约10分钟）
<ol><li>传感器滤芯的水化（步骤时间：约 10 分钟）<ol>
<li>打开细胞外通量测定试剂盒，取下传感器盒和实用板组件。保存装载指南单位，以便第二天使用。</li>
		<li>手动将传感器盒（带盖的顶部绿色部分）与实用板（下部96孔微孔板）分开，并将其倒置放在实用?...

<ol>
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D., Ghaffari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+the+maintenance+of+hematopoietic+stem+cells:+new+perspectives+and+opportunities.">Mitochondria in the maintenance of hematopoietic stem cells: new perspectives and opportunities.</a> Blood. 133 (18), 1943-1952 (2019).</li><li>Inoue, 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=Mitochondrial+respiration+defects+modulate+differentiation+but+not+proliferation+of+hematopoietic+stem+and+progenitor+cells.">Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells.</a> FEBS Letters. 584 (15), 3402-3409 (2010).</li><li>Simsek, T., 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=The+distinct+metabolic+profile+of+hematopoietic+stem+cells+reflects+their+location+in+a+hypoxic+niche.">The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche.</a> Cell Stem Cell. 7 (3), 380-390 (2010).</li><li>Bhatti, J. S., Bhatti, G. K., Reddy, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+oxidative+stress+in+metabolic+disorders+-+A+step+towards+mitochondria+based+therapeutic+strategies.">Mitochondrial dysfunction and oxidative stress in metabolic disorders - A step towards mitochondria based therapeutic strategies.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1863 (5), 1066-1077 (2017).</li><li>Fontenay, M., Cathelin, S., Amiot, M., Gyan, E., Solary, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+hematopoiesis+and+hematological+diseases.">Mitochondria in hematopoiesis and hematological diseases.</a> Oncogene. 25 (34), 4757-4767 (2006).</li><li>Nadanaciva, 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=Assessment+of+drug-induced+mitochondrial+dysfunction+via+altered+cellular+respiration+and+acidification+measured+in+a+96-well+platform.">Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform.</a> Journal of Bioenergetics and Biomembranes. 44 (4), 421-437 (2012).</li><li>Nicholls, D. G., 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=Bioenergetic+profile+experiment+using+C2C12+myoblast+cells.">Bioenergetic profile experiment using C2C12 myoblast cells.</a> Journal of Visualized Experiments: JoVE. (46), e2511 (2010).</li><li>Mookerjee, S. A., Goncalves, R. L. S., Gerencser, A. A., Nicholls, D. G., Brand, 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+contributions+of+respiration+and+glycolysis+to+extracellular+acid+production.">The contributions of respiration and glycolysis to extracellular acid production.</a> Biochimica et Biophysica Acta. 1847 (2), 171-181 (2015).</li><li>Nicholls, D. G., Ferguson, S. J., 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, Fourth Edition. , 255-302 (2013).</li><li>Aft, R. L., Zhang, F. W., Gius, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+2-deoxy-D-glucose+as+a+chemotherapeutic+agent:+mechanism+of+cell+death.">Evaluation of 2-deoxy-D-glucose as a chemotherapeutic agent: mechanism of cell death.</a> British Journal of Cancer. 87 (7), 805-812 (2002).</li><li>Traba, J., Miozzo, P., Akkaya, B., Pierce, S. K., Akkaya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Optimized+protocol+to+analyze+glycolysis+and+mitochondrial+respiration+in+lymphocytes.">An Optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes.</a> Journal of Visualized Experiments: JoVE. (117), e54918 (2016).</li><li>Horan, M. P., Pichaud, N., Ballard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+quantifying+mitochondrial+dysfunction+in+complex+diseases+of+aging.">Review: quantifying mitochondrial dysfunction in complex diseases of aging.</a> The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences. 67 (10), 1022-1035 (2012).</li><li>Morrison, S. J., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+marrow+niche+for+haematopoietic+stem+cells.">The bone marrow niche for haematopoietic stem cells.</a> Nature. 505 (7483), 327-334 (2014).</li></ol>

该方法论文描述了一种优化的方案，用于使用Seahorse细胞外通量分析仪评估小鼠HSPC中的细胞生物能量（糖酵解和OxPHOS）。该装置是一种强大的工具，可同时测量活细胞的ECAR和OCR，分别是糖酵解和线粒体呼吸的指标。因此，它可用于实时评估细胞生物能量学。此外，基于96孔微孔板的平台提供高灵敏度的高通量定量，允许使用单个板同时分析多个样品，而另一种高分辨率呼吸计Oroboros O2k只能同时分?...

造血是由HSCs1形成具有高度专业化功能的各种类型的成熟血细胞的过程。HSC能够自我更新和分化成各种多能和谱系特异性祖细胞群。这些祖细胞最终产生淋巴样、髓系、红细胞和巨核细胞谱系的细胞。为了保持其自我更新能力，HSC在很大程度上保持静止状态，并且与其他组织干细胞一样，被认为依靠糖酵解而不是线粒体OxPHOS来产生ATP2，3。进入细胞周期导致呼吸和OxPHOS增强，导致活性氧（ROS）水平升高，这对HSC的维持和功能有害3。因此，重复的细胞分裂可能导致HSC的自我更新能力降低，并最终导致其耗尽。
在成人造血中，HSC主要经历不对称细胞分裂，在此期间，其中一个子细胞保留HSC潜力并继续依赖糖酵解代谢。另一个子细胞成为原始的祖细胞，失去自我更新能力，但增殖并最终产生分化的功能性造血细胞4。当HSCs分化产生下游祖细胞时，从糖酵解到线粒体代谢的转变被认为是为了提供支持这种快速转变所需的能量和构建块5，正如HSC具有无活性线粒体质量6，7，8，9的观察结果所表明的那样。.相反，线粒体活性（由连锁ROS水平表示）在谱系承诺的祖细胞中远高于HSCs9，10，11。因此，在造血最早阶段发生的代谢变化表明线粒体在调节HSC命运中具有直接和至关重要的作用。
在各种应激条件下（如衰老、癌症、糖尿病和肥胖）下存在的功能失调线粒体12 会干扰 HSC 自我更新能力，通过产生过量的 ROS、损害 ATP 生成和/或改变其他代谢过程来诱导 HSC/祖细胞分化的失衡9，12，13.HSC/祖细胞分化中代谢稳态的扰动可显著影响造血，可能导致血液学异常的发展13.鉴于糖酵解和线粒体OxPHOS对HSC干性和分化的关键影响，研究正常和胁迫条件下的代谢参数是有意义的。通过评估其ECAR和OCR，可以获得对HSC和祖细胞的糖酵解和线粒体功能的宝贵见解，它们分别是细胞糖酵解和线粒体呼吸的指标。
Seahorse细胞外通量分析仪是一种功能强大的仪器，每孔配备两个探针，可同时测量活细胞中的ECAR和OCR，因此可用于实时评估细胞生物能量，以响应各种底物或抑制剂。与分析仪一起使用的检测盒包含进样口，可容纳多达四种药物，以便在测定过程中自动进样。典型的糖酵解压力试验方案如图 1A所示。该测定从测量细胞的ECAR开始，在含有谷氨酰胺但不含有葡萄糖或丙酮酸的糖酵解胁迫测试培养基中孵育。这表示由于细胞的非糖酵解活性而发生的酸化，并且被报道为非糖酵解酸化。接下来以饱和浓度注射葡萄糖。通过糖酵解，细胞中的葡萄糖转化为丙酮酸盐，丙酮酸盐在细胞质中进一步代谢以产生乳酸盐，或在线粒体中产生CO2 和水。
葡萄糖转化为乳酸盐导致净产生并随后将质子释放到细胞外培养基中，导致ECAR14，15，16的快速增加。ECAR中这种葡萄糖刺激的变化被报告为基础条件下的糖酵解。第二次注射由寡霉素（ATP合酶的抑制剂，又名复合物V17）组成，其抑制线粒体ATP的产生。在寡霉素介导的OxPHOS抑制期间，细胞最大限度地上调糖酵解以满足其能量需求。这导致ECAR进一步增加，揭示了细胞的最大糖酵解能力。最大糖酵解能力与基础糖酵解之间的差异称为糖酵解储备。最后，注射2-DG，这导致ECAR显着下降，通常接近非糖酵解酸化水平。2-DG是一种葡萄糖类似物，具有竞争力地与己糖激酶结合，导致糖酵解18的抑制。因此，2-DG诱导的ECAR降低进一步证实糖酵解确实是葡萄糖和寡霉素注射后观察到的ECAR的来源。
图1B 显示了典型线粒体压力测试的示意图。该测定从细胞的基线OCR测量开始，在含有葡萄糖，谷氨酰胺和丙酮酸的线粒体压力测试培养基中孵育。在基础OCR测量之后，在该测定中注入寡霉素，其抑制复合物V，从而减少通过电子传递链（ETC）的电子流动17。因此，OCR在寡霉素注射后降低，OCR的这种降低与线粒体ATP产生有关。第二次注射剂由羰基氰化物-4（三氟甲氧基）苯腙（FCCP），质子团和线粒体OxPHOS17的解偶联剂组成。FCCP通过允许质子流过线粒体内膜来折叠线粒体质子梯度。由于FCCP注射，通过ETC的电子流被抑制，复合物IV以最大水平消耗氧气。最大OCR和基础OCR之间的差异被称为备用呼吸能力，这是细胞在压力条件下对增加的能量需求做出反应的能力的衡量标准。最后，注射两种ETC抑制剂（鱼藤酮，复合物I抑制剂和抗霉素A，复合物III抑制剂17）的混合物，其完全关闭电子流，OCR降低到低水平。鱼藤酮和抗霉素A注射后测量的OCR对应于由细胞内其他过程驱动的非线粒体OCR。非线粒体 OCR 能够计算基底呼吸、质子泄漏和最大呼吸。
基础呼吸代表基线 OCR 和非线粒体 OCR 之间的差异。质子泄漏是指寡霉素注射后的OCR与非线粒体OCR的区别。最大呼吸量表示 FCCP 注射后 OCR 与非线粒体 OCR 之间的差异。耦合效率计算为ATP生产率与基础呼吸速率的百分比。该方法论文提供了使用海马XFe96细胞外通量分析仪测量谱系阴性HSPC中ECAR和OCR的详细方案。该协议描述了分离小鼠谱系阴性HSPC的方法，解释了细胞接种密度和细胞外通量测定中使用的各种药物浓度的优化，并描述了药物治疗策略。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m filter</td>
			<td>Corning</td>
			<td>430626</td>
			<td>Used to filter-sterilize the assay media</td>
		</tr>
		<tr>
			<td>100 mM sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td>Component of mitochondrial stress test assay medium</td>
		</tr>
		<tr>
			<td>15 mL conical Falcon tubes</td>
			<td>Corning</td>
			<td>352096</td>
			<td>Used during HSPCs harvest and to prepare assay drug solutions</td>
		</tr>
		<tr>
			<td>200 mM L-glutamine</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Component of glycolysis stress test and mitochondrial stress test assay media</td>
		</tr>
		<tr>
			<td>2-Deoxy-D-glucose (2-DG)</td>
			<td>Sigma-Aldrich</td>
			<td>D8375</td>
			<td>3rd drug injection during glycolysis stress test</td>
		</tr>
		<tr>
			<td>5x Enrichment buffer (MojoSort)</td>
			<td>Biolegend</td>
			<td>480017</td>
			<td>Used for washings during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Ammonium chloride (NH4Cl)</td>
			<td>Fisher Scientific</td>
			<td>A661-3</td>
			<td>Component of ACK lysis buffer</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>3rd drug injection during mitochondrial stress test</td>
		</tr>
		<tr>
			<td>Bio-Rad DC protein assay kit</td>
			<td>Bio-Rad</td>
			<td>500-0112</td>
			<td>Used as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td>2nd drug injection during mitochondrial stress test</td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>Cell adhesive. Used for coating cell microplates</td>
		</tr>
		<tr>
			<td>Countes 3 Automated Cell Counter</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>O2793-500</td>
			<td>Component of ACK lysis buffer and RIPA lysis buffer</td>
		</tr>
		<tr>
			<td>Falcon 70 &#956;m filter</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td>Used as cells strainer during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Gibco Fetal bovine serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>26400044</td>
			<td>Used to prepare assay buffer during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Gibco HBSS</td>
			<td>Fisher Scientific</td>
			<td>14175095</td>
			<td>Used to prepare assay buffer during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>Component of mitochondrial stress test assay medium and first injection of glycolysis stress test</td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>O4876</td>
			<td>2nd drug injection during glycolysis stress test and 1st drug injection during mitochondrial stress test</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td>Used to wash cells after assay for protein quantification</td>
		</tr>
		<tr>
			<td>Potassium bicarbonate (KHCO3)</td>
			<td>Fisher Scientific</td>
			<td>P235-500</td>
			<td>Component of ACK lysis buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail (PIC)</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>Supplied as tablets. One tablet was dissolved in 10 mL of RIPA buffer to make 1x PIC.</td>
		</tr>
		<tr>
			<td>Rat biotin antimouse-B220, Clone ID: RA3-6B2</td>
			<td>Biolegend</td>
			<td>103203</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Rat biotin antimouse-CD2, Clone ID: RM2-5</td>
			<td>Biolegend</td>
			<td>100103</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Rat biotin antimouse-CD3, Clone ID: 17A2</td>
			<td>Biolegend</td>
			<td>100243</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Rat biotin antimouse-CD5, Clone ID: 53-7.3</td>
			<td>Biolegend</td>
			<td>100603</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Rat biotin antimouse-CD8, Clone ID: 53-6.7</td>
			<td>Biolegend</td>
			<td>100703</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Rat biotin antimouse-Gr-1, Clone ID: RB6-8C5</td>
			<td>Biolegend</td>
			<td>108403</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Rat biotin antimouse-Ter-119, Clone ID: TER-119</td>
			<td>Biolegend</td>
			<td>116203</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td>3rd drug injection during mitochondrial stress test</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 extracellular flux analyzer</td>
			<td>Seahorse Biosciences now Agilent</td>
			<td></td>
			<td>For ECAR and OCR measurments in real time.</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Used to make Cell-adhesive solution for microplate coating</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher</td>
			<td>BP358</td>
			<td>Component of RIPA lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td>Component of RIPA lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium Fluoride (NaF)</td>
			<td>Sigma-Aldrich</td>
			<td>S7920</td>
			<td>Component of RIPA lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td>Prepared 1 N solution. Used for pH normalization</td>
		</tr>
		<tr>
			<td>Streptavidin Nanobeads (MojoSort)</td>
			<td>Biolegend</td>
			<td>480015</td>
			<td>Used for lineage depletion during HSPCs harvest</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher</td>
			<td>BP153</td>
			<td>Component of RIPA lysis buffer</td>
		</tr>
		<tr>
			<td>XF base medium</td>
			<td>Agilent</td>
			<td>102353-100</td>
			<td>base medium used to prepare glycolysis stress test and mitochondrial stress test assay media</td>
		</tr>
		<tr>
			<td>XF prep station</td>
			<td>Seahorse Biosciences</td>
			<td></td>
			<td>Used for non-CO2 37 &#176;C incubations</td>
		</tr>
		<tr>
			<td>XFe96 extracellular FluxPak</td>
			<td>Agilent</td>
			<td>102416-100 or 102601-100</td>
			<td>Includes assay cartridges with utility plates, loading guide flats for loading 
			drugs onto the assay cartridge, XF calibrant solution, and XF cell culture microplate</td>
		</tr>
	</tbody>
</table>

assessment of cellular bioenergetics in mouse hematopoietic stem and primitive progenitor cells using the extracellular flux analyzer

在稳态下，造血干细胞（HSC）在很大程度上保持静止，并且被认为主要依靠糖酵解来满足其能量需求。然而，在感染或失血等应激条件下，HSC增殖并迅速产生下游祖细胞，而下游祖细胞又进一步分化，最终产生成熟的血细胞。在这个转变和分化过程中，HSC从静止状态中退出，并迅速经历从糖酵解到氧化磷酸化（OxPHOS）的代谢转换。各种应激条件，如衰老，癌症，糖尿病和肥胖症，会对线粒体功能产生负面影响，从而改变造血过程中HSC和祖细胞的代谢重编程和分化。通过评估HSC和祖细胞在正常和胁迫条件下的糖酵解和线粒体功能，可以获得对其细胞外酸化率（ECAR）和耗氧率（OCR）的宝贵见解，这分别是细胞糖酵解和线粒体呼吸的指标。 
在这里，提供了一个详细的方案，以使用细胞外通量分析仪测量小鼠骨髓来源的谱系阴性细胞群中的ECAR和OCR，其中包括造血干细胞和原始祖细胞（HSPC）。该协议描述了从小鼠骨髓中分离谱系阴性细胞的方法，解释了这些测定中使用的细胞接种密度和2-脱氧-D-葡萄糖（2-DG，抑制糖酵解的葡萄糖类似物）和各种OxPHOS靶向药物（寡霉素，FCCP，鱼藤酮和抗霉素A）浓度的优化，并描述了药物治疗策略。糖酵解通量的关键参数，如糖酵解、糖酵解容量和糖酵解储备，以及OxPHOS参数，如基础呼吸、最大呼吸、质子泄漏、ATP产生、备用呼吸能力和偶联效率，可以在这些测定中测量。该协议允许在非贴壁性HSPCs上进行ECAR和OCR测量，并且可以推广以优化任何类型悬浮细胞的分析条件。

使用该协议，优化各种OxPHOS靶向药物（用于细胞外通量测定）的细胞数量和浓度，以测量从24周龄雌性C57BL / 6小鼠中分离的HSPC的ECAR和OCR。首先，进行糖酵解胁迫试验，以优化细胞数量和寡霉素浓度。在该测定中使用每孔不同数量的HSPC，范围从5×10 4 到2.5×10 5 。如图 2A 和 图2C所示，非糖酵解酸化速率随着细胞数量的增加而升高，从5×10<...

Watch this Scientific Journal Video about Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer at JoVE.com

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

这里介绍的方法总结了使用细胞外通量分析仪实时测量HSPCs的细胞外酸化速率（ECAR）和耗氧速率（OCR）评估非贴壁小鼠造血干细胞和原始祖细胞（HSPC）中细胞生物能量的优化方案。

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

在造血过程中，造血干细胞经历从糖酵解到氧化磷酸化的代谢转换。该方案可用于评估小鼠造血干细胞和祖细胞的糖酵解和线粒体功能。该方法可用于实时评估细胞生物能量学。基于96孔微孔板的平台提供高通量定量和高灵敏度，允许使用单个板同时分析多个样品。该技术可用于探测化学物质或遗传操作在各种条件下对HSC代谢的影响。它可以帮助阐明维持HSC多能性的代谢途径。虽然目前的研究主要集中在小鼠造血干细胞和祖细胞上，但该方案和这种方法可以很容易地适应任何类型的悬浮细胞的分析条件。在测定前一天，打开细胞外通量测定试剂盒以取出传感器盒和实用板组件。保存装载导轨平底，以备第二天使用。现在，手动将传感器盒与实用板分开，并将其倒置在实用板旁边。使用多通道移液器，用助焊剂测定试剂盒随附的200微升校准剂填充实用板的每个孔，然后将传感器盒放回实用程序板上，确保将传感器完全浸入校准剂中。然后，在手稿中提到的加湿条件下，在37摄氏度的非二氧化碳培养箱中，将实用板与校准剂和组装的传感器盒一起孵育过夜。在测定当天，如文本手稿中所述，每96孔细胞培养微孔板制备2.5毫升细胞粘合剂溶液。打开助焊剂测定试剂盒随附的96孔细胞培养微孔板，并在每个孔的底部分配25微升制备的细胞粘合剂溶液。用盖子盖住微孔板，在室温下在罩内孵育30分钟。孵育后，使用多通道移液器或吸出器除去并丢弃多余的细胞粘合剂溶液，并用200微升无菌超纯水洗涤每个孔两次。将不带盖子的板风干30至45分钟。在室温下将制备的小鼠谱系负性HSPCs以200倍G离心5分钟。丢弃上清液后，将细胞沉淀重悬于适当的测定培养基中，并再次离心细胞悬浮液。再次重复该过程，并将细胞重悬于适当的加热测定培养基中，浓度为每50微升250， 000个细胞或每毫升500万个细胞。使用多通道移液器沿每个孔的细胞粘合剂包被的96孔细胞培养微孔板的侧面加入50微升的细胞悬浮液，然后将50微升的测定培养基加入角背景测量孔中。通过每孔非包被的96孔细胞培养微孔板添加50微升水来创建离心机平衡板。在室温下以200倍G离心细胞一分钟。将板在37摄氏度的非二氧化碳培养箱中孵育25至30分钟以进行细胞附着。30分钟后，在显微镜下目视确认细胞稳定地粘附在微孔板表面。首先在预热的糖酵解胁迫测试测定培养基中制备所有必需的溶液，如文本手稿中所述，并从培养箱中取出水合传感器盒。将传感器盒从校准剂中取出，并将其与校准剂放在同一实用板上，以去除气泡。将 80 装载导轨平放在传感器盒的顶部，使其定位，使字母 A 位于左上角。使用多通道移液器，在端口A中分配20微升100毫摩尔葡萄糖溶液，将80加载导轨平放，将BC加载导轨平放，将字母B定向到左上角。在端口B中分配22微升20微摩尔寡霉素溶液，将BC装载导轨重新定位为平坦，以将字母C定位在左上角，用于装载端口C，并在端口C中分配25微升500毫摩尔2-脱氧-d-葡萄糖溶液，然后取下并丢弃装载导轨平板。现在，在控制器上创建或加载糖酵解压力测试的模板。输入有关注射策略、治疗条件和细胞类型的详细信息，然后按生成组。转到板块图并将孔分配给要分析的每个组。在协议中，确保在初始化步骤中检查校准和平衡。对于基线测量和每次注射后的测量，将测量周期数设置为三次，混合，等待，测量时间为三分钟，零分钟，三分钟。单击开始运行在软件上。从装填的化合物和水合传感器盒中取下盖子，并将其放在细胞外通量分析仪的工作托盘上。开始校准运行。从培养箱中取出含有接种细胞的微孔板。在不干扰细胞的情况下，每孔缓慢加入130微升预热的糖酵解胁迫试验培养基，以将每个孔中的培养基体积补足至180微升，并将板返回到培养箱中再15至20分钟。单击软件上的打开托盘以打开海马的热托盘。校准结束后，用含有细胞的测定微孔板替换实用板，然后按称重传感器板开始测量。测量结束后，在不干扰细胞的情况下从平板中取出测定培养基，并用250微升磷酸盐缓冲盐水轻轻洗涤它们而不会使细胞脱落。现在向每个孔中加入10微升的放射性免疫沉淀裂解缓冲液，补充1X蛋白酶抑制剂鸡尾酒，并在摇摇杯上搅拌平板10分钟。将整个板在零下80摄氏度冷冻。解冻板并按照制造商的说明进行蛋白质测量测定以进行数据归一化。通过使用Wave桌面软件打开数据文件来检索和分析数据。单击“规范化”，粘贴每个孔的规范化值，然后单击“应用”以规范化数据。单击“导出”并选择“糖酵解压力测试报告生成器”，将分析的数据导出到报告生成器。非糖酵解酸化速率随着细胞数量的增加而上升，从50， 000增加到250， 000，但在200， 000到250， 000之间仅略有增加。注射10毫摩尔葡萄糖刺激所有细胞数量的糖酵解，在每孔250， 000个细胞时观察到ECAR的最大增加。然而，注射两微摩尔寡霉素不会进一步增加ECAR。在同一组糖酵解胁迫试验中获得的OCR数据显示，由于复合物5抑制，寡霉素注射后显着下降。计算糖酵解胁迫试验参数，选择2微摩尔寡霉素中每孔250， 000个细胞进行进一步研究。线粒体负荷试验表明，两次微摩尔寡霉素注射通过抑制复合物5引起OCR的显着降低。FCCP以剂量依赖性方式刺激HSPCs中的OCR，在两个微摩尔处观察到最大增加。0.5微摩尔鱼藤酮和0.5微摩尔抗霉素A注射液的混合物将OCR降低到与非线粒体耗氧量相对应的最小水平。计算线粒体负荷试验参数，选取2微摩尔FCCP进行进一步研究。鉴于该技术的高通量性质，此处描述的方案可以很容易地适应于筛选造血干细胞，造血祖细胞和恶性造血细胞中的大量生物能量调节剂。海马分析在文献中被广泛用于在各种底物和抑制剂存在下测量各种情况下的新陈代谢。

Research

JoVE Journal

Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

分离和造血干细胞移植（造血干细胞）

科研

生物学

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

小鼠原发性肾管上皮细胞的分离、鉴定及高通量的细胞外流量分析

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

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 ...