Jan Van den Bossche received a Junior Postdoc grant from the Netherlands Heart Foundation (2013T003) and a VENI grant from ZonMW (91615052). Menno de Winther is an Established Investigator of the Netherlands Heart Foundation (2007T067), is supported by a Netherlands Heart Foundation grant (2010B022) and holds an AMC-fellowship. We also acknowledge the support from the Netherlands Cardiovascular Research Initiative, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development and the Royal Netherlands Academy of Sciences for the GENIUS project &#8220;Generating the best evidence-based pharmaceutical targets for atherosclerosis&#8221; (CVON2011-19). We acknowledge Seahorse Bioscience for making open access publication possible. The authors thank Riekelt Houtkooper and Vincent de Boer (Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands) for all previous assistance with the Seahorse analyzer.

1.培养和偏光骨髓来源的巨噬细胞<ol><li>第0天:分离股骨和胫骨来自感兴趣6-10周龄小鼠（在此测定之C57B1 / 6）的骨头。 </li><li>从骨骼去除肌肉组织和放置的骨头一个9厘米培养皿装有冰冷​​的PBS。 </li><li>转移骨头到9厘米的培养皿填充有70％乙醇，轻轻摇动平板30秒。 </li><li>转移骨头到一个新的9厘米的培养皿装有冰冷​​无菌PBS。 </li><li>切股骨和胫骨的两端，用无菌剪刀。 </li><li>使用10毫升注射器和一个地下25针冲洗骨用10毫升冰冷的无菌PBS。收集骨髓在50ml管中。 </li><li>使用23政针转移的骨髓细胞到新的50ml管中。 </li><li>重复此步骤，以将25g针头。注:需要这些步骤以去除细胞团块和骨骼的残留物。 </li><li>离心机在300×g离心5分钟，在4℃。 </li><li>丢弃SUPernatant和重悬细胞于40ml细胞培养基（RPMI-1640加2mM的L-谷氨酰胺，10％FCS，青霉素（100U /毫升），链霉素（100微克/毫升）和15％过滤的L- 929细胞（ ATCC，CCL-1）含有L- 929细胞条件培养基的M-CSF（或备选地为10ng / ml的重组M-CSF代替-conditioned介质）。 注释:L-929细胞条件培养基的一代人以前在该杂志上详细描述了在2013年11 </li><li>培养从一只小鼠在37℃，5％CO 2培养箱中的细胞两个15厘米的培养皿（不使用细胞培养处理板，因为巨噬细胞将不会从该塑料分离）以每板20 ml的培养基中。 </li><li>加入10 mL的新鲜细胞培养基在第3天，不除去20个ml的较老的细胞培养基。 </li><li>与在第6天20个ml的新鲜培养基这样做更换各板的旧培养基，非贴壁细胞也将被删除。 </li><li>第8天: <ol><li>通过将它们5米分离细胞在在37℃下在10毫升柠檬酸盐盐水（135毫氯化钾加15mM柠檬酸钠在H 2 O的，高压灭菌），并用10毫升的PBS。计数使用细胞计数器成熟骨髓衍生的巨噬细胞（BMDM）在第8天。 </li><li>通过目测或用流式细胞仪验证形成成熟的（细胞CD11b + F4 / 80 +）BMDM。块10 5个细胞与1/100抗CD16 / CD32的在PBS中20分钟在100μlPBS中，孵育1/200抗CD11b-FITC加1/200抗F4 / 80-APC-Cy7的在RT，洗并通过流式细胞仪分析。 注:培养小鼠骨髓来源的巨噬细胞已经发表在细节之前在本杂志由英等 [11]。 </li></ol></li><li>种子50000细胞（最佳细胞计数需要优化）在细胞培养微板在100μl培养基中每孔。不要种子细胞背景校正井（A1，A12，H1，H12），只有把培养基（无细胞）在这些井中提示:按住吸管在一个角度一个回合中间各孔中最齐的细胞层的一侧。 </li><li>使细胞在室温定居在细胞培养罩1小时。这将促进甚至细胞分布，减少边缘效应。使用显微镜和培养在37℃，5％CO 2培养箱监视粘附。 </li><li>三小时后镀，刺激细胞24小时，用10ng / ml的LPS，或20纳克/毫升的重组小鼠IL-4，以产生M1或M2巨噬细胞，分别。包括未处理的幼稚（M0）的巨噬细胞作为对照。通过测量IL-6，IL-12的LPS诱导的分泌评估适当的M1和M2巨噬细胞极化，肿瘤坏死因子（ELISA）和IL-4诱导精氨酸酶1活性和/或MMR（CD206）和MGL（CD301）表面表达（流式细胞仪）如前面3,11描述。 注意:在这一点上，细胞可以是（预）处理的与感兴趣或巨噬细胞可与其他因素来刺激药理化合物。因为，不同孔之间的差异可能是潜艇tantial，最好是每种条件使用至少4，理想的6个孔。 </li></ol> 2.准备胞外通量测定法<ol><li>在运行试验前一日水合传感器盒: <ol><li>将传感器盒倒挂旁边的工具盘。 </li><li>填充效用板用200μl的校准溶液的各孔中，并降低传感器盒上的效用板浸没传感器在校准溶液。 </li><li>验证校准解决方案的水平高到足以保持淹没传感器和发生在非CO 2 37℃培养箱O / N。 </li></ol></li><li>制备测定培养基上测定当天如下: <ol><li>温馨50毫升XF基中期到37℃。 </li><li>加入500微升200mM的L-谷氨酰胺得到2mM的终浓度。 </li><li>用1N的NaOH调节pH至7.4和消毒用0.2微米过滤器，并保持在测定培养基中于37℃。 </li></ol></li><li>轻轻地从偏振巨噬细胞除去细胞培养基，并储存在-20℃以供将来使用（例如，ELISA法来检查正确的巨噬细胞极化）。洗涤细胞用100微升试验中，每孔加入180微升检测培养基和细胞没有冲走在显微镜下检验。放置细胞培养板在37℃培养箱无CO 2的事先到测定运行1小时。 </li><li>准备10×注射复方混合物A到ð 如表1所示 ，暖至37℃，调节pH值至7.4，过滤消毒。 <ol><li>使在这些混合物中的所有化合物在10倍的细胞培养孔中的最终浓度和优化这些浓度对于每个细胞类型。 </li></ol></li><li>使用所提供的装载导板与在端口A，B，C和D，respe制备的10倍的喷射化合物的混合物的体积的指示（表1）装载传感器盒沉着应对。 </li></ol><table border="0"><tbody><tr><td> 混合/注塑 </td><td> 化合物 </td><td> 添加量来获得10倍的混合物（微升） </td><td> 体积测定培养基（毫升） </td><td> 卷运行过程中注入（微升） </td><td> 最终浓度在测定 </td></tr><tr><td>一个</td><td> 2.5M的（45％）的葡萄糖</td><td> 300 </td><td> 2.7 </td><td> 20 </td><td> 25毫米</td></tr><tr><td>乙</td><td> 5毫米寡A（OM） </td><td> 9 </td><td> 3.0 </td><td> 22 </td><td> 1.5微米</td></tr><tr><td> C </td><td> 5毫米FCCP </td><td> 9 </td><td> 2.7 </td><td> 25 </td><td> 1.5微米</td></tr><tr><td></td><td> 100丙酮酸钠溶液</td><td> 300 </td><td></td><td></td><td> 1毫米</td></tr><tr><td> ð </td><td> 5毫米抗霉素A（AA） </tD&gt; <td> 15 </td><td> 3.0 </td><td> 28 </td><td> 2.5μM </td></tr><tr><td></td><td> 5毫米鱼藤酮（ROT） </td><td> 7.5 </td><td></td><td></td><td> 1.25μM </td></tr></tbody></table> 表1.注射液的混合物。 偏光M1和M2的BMDM使用外流量分析仪3.生物能表征<ol><li>创建与2分钟MIX和3分钟的车MEASURE倍和（至少）3混合并率测量试验向导的测定模板各4次注射之前（ 表1）和最后一次注射后循环。 注意:这些浓度是有效的骨髓来源的巨噬细胞和巨噬细胞的Raw264.7细胞系，但对于每种细胞类型应优化。在FCCP混合物加入丙酮酸是需要的燃料的最大呼吸。 </li><li>通过按启动底部开始运行，并按照设备的说明书。首先加载车盒盘与水合探针，以允许通过设备和下一个负荷单元板校准。 </li><li>在测定后，小心地丢弃所有的测定培养基并于-20存储所分析的细胞培养微板℃，使用细胞增殖测定试剂盒由供应商中详细描述的未来细胞正常化。 <ol><li>简要地说，制备1×细胞裂解缓冲液通过在蒸馏水中稀释的组分B的20倍，并稀释化合物A 400倍，在1×细胞裂解缓冲液。加入200μl每孔，孵化5分钟，在室温下，测量荧光〜180 nm激发和〜520 nm的最大发射。 注意:当与非贴壁细胞的工作，或者如果依从性差是一个问题，直接的细胞增殖试剂盒可作为一种替代，因为这个协议不需要丢弃所有测定培养基。然而，比起在本实验中使用的协议，这直接协议是稍微不那么敏感。 </li></ol></li><li>荧光测量后，恢复正常在每个细胞计数以及一个比，其中所有幼稚细胞巨噬细胞的孔的平均细胞数被设定为1。 注:蛋白质定量（例如使用BCA测定）可以作为标准化的细胞计数的另一种方法。然而，在我们手中相比的细胞增殖试验试剂盒，该测定是较不敏感。 </li></ol>

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我们实验室的目的是了解如何巨噬细胞极化和功能可以与最终目标，以改善动脉粥样硬化和其它炎性病症12的结果的影响。为了评估偏振巨噬细胞，人们通常测量LPS（+IFNγ）诱导和IL-4诱导的M1和M2的标记基因（定量PCR）的基因表达，确定IL-6，IL-12，TNF和分泌NO（通过ELISA和格里斯反应），检验IL-4诱导的CD71，CD206，CD273和CD301的表面表达（通过流式细胞术）和精氨酸酶的活性3,13。此外，细胞凋亡测定（通过膜联蛋白V / PI染色），吞噬作用测定法（用荧光胶乳珠和/或pHrodo 大肠杆菌 ）和泡沫细胞形成测定法（由的DiI-脂蛋白摄取，LipidTOX中性脂质染色）可被执行以评估巨噬细胞的功能14。此外，细胞外通量分析可以被执行以测量生物能极化的mac型材噬细胞作为一种新的和替代的功能读出。 这份手稿表明，巨噬细胞极化导致代谢重新编程与LPS诱导巨噬细胞切换到糖酵解增加它们的能量来源。相反，IL-4诱导的巨噬细胞的M2使用线粒体氧化磷酸作为ATP的主要来源。重要的是，代谢重新编程不仅提供能量的M1和M2极化巨噬细胞的特殊要求。事实上，代谢变化影响代谢物浓度是巨噬细胞的表型10,15,16的直接监管。因此，巨噬细胞的新陈代谢，不仅是特性（如图这里在图2中），而且也鲜明的巨噬细胞的偏振状态的驱动器，这新的知识支持immunometabolism研究领域的快速发展，在过去的几年。 然而，在宏代谢概况健壮测量噬菌体依然严峻，有其局限性。在这项研究中，细胞外磁通分析器被施加到鲁棒测量糖酵解，糖酵解储备，最大糖酵解容量，非糖酵解酸化，基底和最大呼吸，ATP产生，备用呼吸容量，质子泄漏和非线粒体呼吸实时在巨噬细胞的一个最小量。 因此，我们修改和改进制造商的协议如下。标准美图压力测试（＃103015-100）建议使用中葡萄糖和丙酮酸和3次注射（OM，FCCP，AA / ROT）的协议。我们选择开始与葡萄糖/丙酮酸的培养基的测定中，添加葡萄糖作为第一注射口A（象在糖酵解压力测试＃103020-100）中，用所述FCCP解偶联剂在端口C以这种方式添加丙酮酸一起，所有相关的糖酵解参数从ECAR测量1-9和有关mitochondri所有相关信息来源人的功能从OCR测量4-15。 需要注意的是它为丙酮酸连同FCCP注入后解偶联到燃料最大呼吸是至关重要的。使用这种组合的协议之前，人们也应该验证-2-脱氧-D-葡萄糖（2-DG）降低该测定的基础值（1-3）后ECAR水平，且录制ECAR率4-6是确因以糖酵解。此外，应该理解，在该手稿中使用的化合物的浓度和细胞数是有效的骨髓来源的巨噬细胞和巨噬细胞的Raw264.7细胞系和其它细胞类型应优化。而且，应该注意的是M-CSF，用来产生骨髓来源的巨噬细胞促进一个M2状抗炎表型。因此所呈现的结果从骨髓衍生的巨噬细胞可以是不完全与用主组织驻留的巨噬细胞或巨噬细胞的细胞系的测量不期间细胞culturi需要M-CSFNG。 相比于现有的方法，该协议要求的巨噬细胞的最小量，并迅速提供了几乎所有的基础代谢细胞特征。这导致足够剩余细胞以执行其它免疫学测定法，甚至用于生物能量谱的细胞仍然可以用于其它的测定后旨意。此外，特定的96孔板格式允许评估实时多个条件在同一时间。然而，各个孔之间的变化可以是相当大的，因此，每个条件使用至少4个孔的通常需要。采取这种限制考虑在内，所描述的外磁通分析仍然允许运行超过10的实验条件（6）一次，这比传统技术更。 总体而言，本文提供了一个简单的和可重复的功能测定，使测量所有相关的糖酵解和线粒体参数ETERS在极化巨噬细胞亚群。这种技术可以作为未来应用以评估巨噬细胞功能，并评价不同的操作上的巨噬细胞的（代谢）的表型的影响 （例如基因敲除，新药物等 ）。因此，细胞外通量数据可以结合使用其他测定，以允许深入表征的巨噬细胞激活状态。

虽然巨噬细胞发挥在几乎所有的疾病中发挥核心作用，调节其表型的分子机制还没有完全拆开的。巨噬细胞显示出高的异质性，并响应于微环境采用不同的表型1。 LPS（+IFNγ）诱导激活经典的M1或M（LPS），巨噬细胞促进炎症，并防止不同类型的微生物威胁2的主机。其他使用IFNγ单独或与肿瘤坏死因子的刺激诱发M1两极分化。 IL-4和/或IL-13诱导的替代巨噬细胞活化和这些M 2 或M（IL-4）细胞是有效的抑制剂和正在进行的免疫反应3控制器。偏光巨噬细胞显示细胞代谢的独特调控脂多糖激活的巨噬细胞M1进行代谢开关增强糖酵解4-6。相反，增强脂肪酸氧化（FAO）和线粒体oxidativë磷酸化（OXPHOS）提供持续的能量中的IL-4诱导的巨噬细胞的M2 7-9。因此，改变的代谢不仅是偏振光巨噬细胞子集的特性的同时，还进行适当的极化和炎症调节的先决条件。重要的是，糖酵解或OXPHOS的抑制/ FAO已经证明削弱M1或M2激活，分别8,10。这样，在巨噬细胞识别代谢变化可作为一种工具，以评估它们的极化状态和炎性电位施加。 一致的测定法，其测量细胞巨噬细胞代谢因此可以用于预测药物，基因是否击倒或其他处理的影响巨噬细胞极化和功能。在此视频，一个细胞外通量分析仪用于表征幼稚，M1和M2巨噬细胞的生物能学概况。 这份手稿详细介绍了优化的协议，允许测量所有相关的参数酵解（糖酵解，最大糖酵解和糖的储备）和线粒体功能特点（共呼吸，基础线粒体呼吸，ATP的产生，质子漏，最大呼吸和备用呼吸能力）在一个单一的检测。使用该实验装置，生物能可以控制和&#39;改变&#39;之间进行比较（ 如基因敲除，转基因过表达或药物治疗）的细胞。

<table border="0">
	<tbody>
		<tr>
			<td>23G and 25G needles</td>
			<td>Becton Dickinson</td>
			<td>#300800 and #300600</td>
			<td>To flushed bone marrow</td>
		</tr>
		<tr>
			<td>10 ml syringes</td>
			<td>Becton Dickinson</td>
			<td>#307736</td>
			<td>To flushed bone marrow</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Greiner</td>
			<td>#639161</td>
			<td>To culture bone marrow cells</td>
		</tr>
		<tr>
			<td>CyQUANT Cell Proliferation Assay kit&#160;</td>
			<td>Molecular Probes</td>
			<td>#C7026</td>
			<td>For cell quantification at a later time point (works only for adherent cells)</td>
		</tr>
		<tr>
			<td>CyQUANT Direct Cell Proliferation Assay kit&#160;</td>
			<td>Molecular Probes</td>
			<td>#35011</td>
			<td>For cell quantification immediately after assay (non-adherent cells)</td>
		</tr>
		<tr>
			<td>XFe96 cell culture microplate</td>
			<td>Seahorse Bioscience</td>
			<td>#101085-004</td>
			<td>96 well plate in which cells are cultured and in which assay is done</td>
		</tr>
		<tr>
			<td>recombinant mouse interleukin-4 (IL-4)</td>
			<td>Peprotech</td>
			<td>#214-14-B</td>
			<td>Used to induced alternative macrophage activation</td>
		</tr>
		<tr>
			<td>lipopolysaccharide (LPS)</td>
			<td>Sigma</td>
			<td>#L2637</td>
			<td>Pro-inflammatory stimulus</td>
		</tr>
		<tr>
			<td>Seahorse Sensor Cartridge&#160;</td>
			<td>Seahorse Bioscience</td>
			<td>#102416-100</td>
			<td>Contains the probes for pH and O2 measurement</td>
		</tr>
		<tr>
			<td>Seahorse XF Calibrant&#160;</td>
			<td>Seahorse Bioscience</td>
			<td>#100840-000</td>
			<td>Needed to hydrate the probes overnight&#160;</td>
		</tr>
		<tr>
			<td>XF base medium&#160;</td>
			<td>Seahorse Bioscience</td>
			<td>#102353-100</td>
			<td>Buffer-free medium that allows efficient measurement of pH changes</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life Technologies</td>
			<td>#25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>#G8769</td>
			<td>Fuels glycolysis once added to the cells</td>
		</tr>
		<tr>
			<td>oligomycin A</td>
			<td>Sigma</td>
			<td>#75351</td>
			<td>Inhibits mitochondrial ATP synthase</td>
		</tr>
		<tr>
			<td>FCCP( Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)</td>
			<td>Sigma</td>
			<td>#C2920</td>
			<td>Uncouples mitochondrial respiration</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate solution</td>
			<td>Lonza</td>
			<td>#BE13-115E</td>
			<td>Allows maximal mitochondrial respiration without the need for glycolysis</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma</td>
			<td>#BE13-115EA8674</td>
			<td>Inhibits complex III of the mitochondria</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>#BE13-115EA8674R8875</td>
			<td>Inhibits complex I of the mitochondria</td>
		</tr>
		<tr>
			<td>Casy Cell Counter</td>
			<td>Roche Diagnostics</td>
			<td>#05 651 697 001</td>
			<td>Instrument to count cells</td>
		</tr>
		<tr>
			<td>anti-CD16/CD32</td>
			<td>eBioscience</td>
			<td>#14-0161</td>
			<td>Fc receptor block flow cytometry</td>
		</tr>
		<tr>
			<td>anti-F4/80-APC-eFluor780</td>
			<td>eBioscience</td>
			<td>#47-4801</td>
			<td>Antibody to stain macrophages</td>
		</tr>
		<tr>
			<td>anti-CD11b-FITC</td>
			<td>eBioscience</td>
			<td>#11-0112</td>
			<td>Antibody to stain macrophages</td>
		</tr>
	</tbody>
</table>

metabolic characterization of polarized m1 and m2 bone marrow-derived macrophages using real-time extracellular flux analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

在完成细胞外通量测定法和细胞定量后，数据可以标准化用于细胞计数和分析。这通常产生ECAR和OCR曲线，如图1A和图1B。 注射葡萄糖（A）后，增加ECAR代表糖酵解率。 ATP合酶抑制与寡（B）的后ECAR额外增加提供有关糖酵解储备和能力（图1A）的信息。在分析OCR值，寡注射液（B）可用于线粒体ATP合成耗氧量的计算。 FCCP（C）脱开线粒体呼吸和相应的OCR测量产生有关的最大和备用呼吸容量的数据。最后，注射鱼藤酮（ROT）和抗霉素A（AA）的方框线粒体复合物I和III和残留的OCR表示非线粒体氧利弊umption（图1B）。 <img alt="图1" src="/files/ftp_upload/53424/53424fig1.jpg" /> 图1:来自胞外XF通量测定法衍生的代谢参数 。 （ 一）细胞糖酵解以下参数从ECAR值计算（在MPH /分钟）:糖酵解，最大糖酵解能力和糖酵解储备。 （B）中的OCR测量（在皮摩尔/分钟）来计算线粒体功能的下一个基本参数:基础呼吸，ATP产生，质子泄漏，最大呼吸和备用呼吸容量。胰高血糖=葡萄糖; OM =寡A; FCCP =羰基氰化物4-（三氟甲氧基）苯腙; AA =抗霉素A;腐=鱼藤酮<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53424/53424fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 运行后，ECAR和OCR测量1直到升15为每个孔可以被导出到Excel和下面的糖酵解参数可以计算从ECAR测量如下: 非糖酵解酸化=平均。 ECAR（1,2,3） 糖酵解=平均。 ECAR（4,5,6） -魅力。 ECAR（1,2,3） 最大糖酵解能力=平均。 ECAR（7,8,9） -魅力。 ECAR（1,2,3） 糖酵解储备=平均。 ECAR（7,8,9） -魅力。 ECAR（4,5,6） 从OCR率，下代谢特征可以被确定: 非线粒体呼吸=平均。 OCR（13,14,15） 基础呼吸=平均。 OCR（4,5,6） -魅力。 OCR（13,14,15） ATP的产生=平均。 OCR（4,5,6） -魅力。 OCR（7,8,9） 质子漏=平均。 OCR（7,8,9） -魅力。 OCR（13,14,15） 最大呼吸=平均。 OCR（10,11,12） -魅力。 OCR（13,14,15） 备用呼吸能力（SRC）=平均。 OCR（10,11,12） -魅力。 OCR（4,5,6） <img alt="图2" src="/files/ftp_upload/53424/53424fig2.jpg" /> 图2:对幼稚代谢特性（M0），LPS-（M1）和IL-4-（M2），巨噬细胞偏振光对于每次测量的8各个孔的平均值（SEM）的，平均值和标准误差呈现。 （A）的细胞外酸化速率（ECAR，在MPH /分钟）和（B）的氧消耗速率（OCR，在皮摩尔/分钟），测量用于差动极化（M1和M2）和幼稚的巨噬细胞（M0）。所有测量均标准化为细胞计数（与CyQUANT测量），幼稚的巨噬细胞的平均细胞数设定为1。jection A =葡萄糖; B =寡; C = FCCP; D =抗霉素A +鱼藤酮。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53424/53424fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 如图2A所示，巨噬细胞活化与LPS诱导提高糖酵解代谢。当看耗氧率（OCR） 在图2B中的LPS和IL-4处理的巨噬细胞之间的差异更加明显。实际上，虽然最大氧化代谢是高度抑制在M（LPS），IL-4诱导基底和尤其最大呼吸中的M2巨噬细胞。应该注意的是增加糖酵解是相当独特的，以M（LPS）的巨噬细胞，而且也不一定M1型巨噬的特征，因为M（IFNγ）的巨噬细胞不显示提高糖酵解。 总体来说，这款外流量分析显示，塔吨的IL-4诱导的巨噬细胞的M2的特征在于增强的氧化代谢和特别高的备用呼吸容量（SRC），而脂多糖活化巨噬细胞显示增强的糖酵解代谢和不具有任何备用呼吸容量。

Watch this Scientific Journal Video about Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis at JoVE.com

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

metabolic-characterization-polarized-m1-m2-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

35.2K 次观看.  Academic Medical Center, Amsterdam. Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage’s metabolism and phenotype.

视频: Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

使用实时细胞分析监测宿主外的流感病毒存活率

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-&#947; and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4- or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong up-regulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1-polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected and Mtb-infected cells at 24-hours post-infection. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs in different areas of research.

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

This protocol provides a method to study Mycobacterium tuberculosis infection in human M1- or M2-polarized macrophages based on differentiation of peripheral-blood-monocytes to macrophage-like cells that are infected with the GFP-labeled virulent strain H37Rv, and analyzed with flow cytometry using a 10-color panel including expression of selected M1/M2 markers.

M1 和 M2 人类单细胞衍生细胞的极化和对 结核病 感染的流动细胞学分析

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

骨髓来源巨噬细胞的分离和 体外 培养用于NO-氧化还原生物学研究

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

摘要

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此视频中的章节

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

使用骨髓源性巨噬细胞巨噬细胞极化的调查

骨髓衍生的巨噬细胞产

隔离和小鼠骨髓的静脉注射单核细胞衍生

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

使用实时细胞分析监测宿主外的流感病毒存活率

M1 和 M2 人类单细胞衍生细胞的极化和对结核病感染的流动细胞学分析

骨髓来源巨噬细胞的分离和体外培养用于NO-氧化还原生物学研究

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

摘要

探索更多视频

此视频中的章节

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

使用骨髓源性巨噬细胞巨噬细胞极化的调查

骨髓衍生的巨噬细胞产

隔离和小鼠骨髓的静脉注射单核细胞衍生

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

使用实时细胞分析监测宿主外的流感病毒存活率

M1 和 M2 人类单细胞衍生细胞的极化和对 结核病 感染的流动细胞学分析

骨髓来源巨噬细胞的分离和 体外 培养用于NO-氧化还原生物学研究

M1 和 M2 人类单细胞衍生细胞的极化和对结核病感染的流动细胞学分析

骨髓来源巨噬细胞的分离和体外培养用于NO-氧化还原生物学研究