支持这项工作是由美国心脏协会（BGIA 7850037周北雁博士）。

1。骨髓细胞的分离<ol><li>隔离6-8周龄小鼠的股骨和胫骨，用清水冲洗干净的头发和然后剖开骨头。 </li><li>使用21G针和10毫升注射器，冲洗出来的骨髓成冷PBS +2％热灭活胎牛血清（FBS）（3-5毫升/鼠标）。 </li><li>通过21G针4-6次通过骨髓细胞分解。 </li><li>通过细胞通过一个70微米的细胞过滤去除细胞团块，骨骼，头发和其他细胞/组织。 </li><li>加入3体积的NH 4 Cl溶液（0.8％NH 4 Cl溶液，干细胞技术），并在冰上孵育10分钟以除去红血细胞。 </li><li>降速细胞在500 XG 5分钟，在4°C。 </li><li>在冷PBS +2％胎牛血清（20-50毫升，根据细胞的数量），重悬细胞沉淀。 </li></ol> 2。感应BMDM形成<ol><li>重悬隔离BMDM生长培养基中的骨髓细胞（2×10 6个细胞/米升）。 </li></ol> BMDM生长培养基: Iscove氏改性的Dulbecco的中等（IMDM）+ 10％FBS + 15％过滤（0.2微米）ł-929细胞（ATCC CCL-1）培养上清液（含有单核细胞集落刺激因子，M-CSF）或10纳克/毫升中号-CSF。 注意:L-929细胞上清液中包含M-CSF 18。 ，以确保有效的活性的条件培养基中，5×10 5 L-929细胞接种在T75厘米2烧瓶中6-7天，收集条件培养基，并通过0.45微米过滤器在使用前。介质的等分试样，可立即使用或储存在-80℃下1-2个月。 <ol start="2"><li>在6或12孔组织培养板（根据实验设计）（科宁Costar）中的种子细胞。 </li><li>变更新鲜BMDM生长培养基，在第3天。 </li><li> 在第7天，形成成熟的BMDM使用流式细胞分析评价荧光标记的抗体检测细胞表达CD11b和F4/80的。 </li></ol> 3。 BMDM极化激活<ol><li>在第7天，改变新鲜刺激培养基:M1激活 ，用IMDM培养基含10％FBS和100 ng / ml的LPS或100 ng / ml的LPS与50毫微克/毫升IFNγ;为M2活化 ，使用含有10％FBS的IMDM与10 ng / ml的IL-4和/或10 ng / ml的IL-13。 </li><li>收藏报错受激BMDMs的使用温暖的0.05％胰蛋白酶从盘分离，其次是含有10％FBS的PBS洗涤细胞两次。 </li></ol> 注意:要分离，重悬后分化成熟的巨噬细胞，0.05％胰蛋白酶溶液（含有0.48 mM的EDTA，Invitrogen公司）或2-5 mM的EDTA钙和Mg的PBS或Hank氏平衡缓冲液（HBSS）中，都可以使用。当使用含胰蛋白酶消化培养基，细胞在37℃下处理小于10分钟，以避免丢失河畔脸蛋白由于过度消化。 <ol start="3"><li>使用抗体检测细胞表面抗原的表达，包括CD11b和F4/80的CD11c，CD206，CD69，CD80或CD86在不同时间点，使用标准的流式细胞仪染色程序。 </li><li>确定M1和M2的巨噬细胞活化的特征，包括IL-1β，TNF-α和IL-6（M1激活）或IL-10，IL-13，精氨酸酵素和PPARγ（M2活化）使用定量RT-PCR的基因的表达。确定激活参与细胞信号转导通路激活M1或M2巨噬细胞的免疫印迹分析。 </li></ol>

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没有利益冲突的声明。

我们在这里报告一个简单而诱导激活巨噬细胞来源于骨髓祖细胞在体外容易适应的过程。此过程可用于调查负责的巨噬细胞极化机制。成熟的巨噬细胞获得使用该协议平均为95％至99％的纯度，需要没有额外的纯化程序。要调查的特定基因的功能利益在巨噬细胞的极化，异位表达或基因特异性敲除的上下文中，可以进行以下的转染的细胞在第7天。此协议也将提供一个7天的培养窗口中的某些因素或影响形成，成熟和巨噬细胞的表型的基因的影响进行调查。 活化的巨噬细胞显示复杂的细胞和分子公司，具有很大的可塑性，响应各种刺激3，9，19。为例如，LPS诱发有效的亲炎症M1响应，可以进一步增强在IFNγ或肿瘤坏死因子（TNF）和IL-4和IL-13都刺激M2激活的存在下，但是，不完全重叠激活的档案3，4，9，15，19，20。在本协议中的结果仅代表了典型的实验结果与骨髓巨噬细胞通过流式细胞仪，荧光定量RT-PCR和免疫印迹分析。表面抗原呈递和相关的细胞信号转导通路激活巨噬细胞不同注意到大量文献对巨噬细胞极化1-8。

有别于经典的炎症反应，巨噬细胞浸润组织通常显示的极化激活状态，起着至关重要的作用，在调节宿主组织的生理功能1-8。刺激后，巨噬细胞活化可以分为经典（M1）和替代（M2）激活2，4，9。 M1巨噬细胞活化取决于Toll样受体（TLRs）和激活核因子κB（NFκB）/ C-Jun氨基末端激酶1（JNK1），导致生产的炎性细胞因子，如TNF-α和IL- 1β，诱导型一氧化氮合酶的激活，导致产量增加的活性氧物种，如氮化氧化氮（NO）10，11。与此相反，M2巨噬细胞的招募的活化PPARγ，PPARδ，或IL-4-STAT6通路，导致替代，消炎（M2）的激活与甘露糖受体上调CD206和精氨酸酶1（Arg1的）6，12 - 14 &lt;/ SUP&gt;。 目前骨髓衍生的巨噬细胞（BMDM），的一个理想的体外模型中了解的机制，活化的巨噬细胞极化控制15。具体而言，M1巨噬细胞的激活可以由脂多糖（LPS）的刺激被诱导，而M2巨噬细胞，可诱导的IL-4和/或IL-13的偏振。成熟骨髓来源的巨噬细胞和活化的巨噬细胞，通过流式细胞术分析，可确定表面抗原的表达，包括F4/80的细胞CD11b，CD11c的，CD206，CD69，CD80和CD86的9，16，17。此外，在细胞因子的产生和细胞与巨噬细胞的极化相关的信号转导通路的变化可以测量通过定量RT-PCR和免疫印迹分别。总之，小鼠骨髓来源的巨噬细胞可以作为一个相关的模型来研究在体外巨噬细胞的极化。

<table><tbody><tr><td>IMDM</td><td>Thermo Scientific</td><td>SH30259.01</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen</td><td>10438-026</td><td></td></tr><tr><td>Murine GM-CSF</td><td>PeproTech</td><td>315-03</td><td></td></tr><tr><td>NH4Cl</td><td>StemCell Technologies</td><td>7850</td><td></td></tr><tr><td>L-929</td><td>ATCC</td><td>CCL-1</td><td></td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>10 x PBS</td><td>Thermo Scientific</td><td>AP-9009-10</td><td></td></tr><tr><td>Anti-mouse CD11b-APC</td><td>eBioscience</td><td>17-0112-81</td><td></td></tr><tr><td>Anti-mouse F4/80-FITC</td><td>eBioscience</td><td>11-4801-81</td><td></td></tr><tr><td>Anti-mouse CD69-PE</td><td>eBioscience</td><td>12-0691-81</td><td></td></tr><tr><td>Anti-mouse CD86-PE</td><td>eBioscience</td><td>12-0862-81</td><td></td></tr><tr><td>Propidium Iodine</td><td>Invitrogen</td><td>P3566</td><td></td></tr></tbody></table>

investigation of macrophage polarization using bone marrow derived macrophages

本文介绍了一种容易容易适应体外模型研究巨噬细胞极化。在存在下GM-CSF/M-CSF，从骨髓中的造血干/祖细胞定向分化成单核细胞，由M1或M2的刺激。激活状态可以跟踪的细胞表面抗原，基因表达和细胞信号转导通路的变化。

BMDM生成过程的示意说明，提出了（ 图1）。在第7天的CD11b + F4/80 +细胞占95％〜99％（ 图2）时，可以观察到成熟的巨噬细胞的高纯度。可以检查使用抗CD11b抗体，F4/80偏光巨噬细胞，CD11c和CD206随​​后通过流式细胞仪分析。 如图3所示，被检测为M1巨噬细胞细胞CD11b + F4/80 + CD11c +细胞CD206-细胞（Q2），而M2巨噬细胞细胞CD11b + F4/80 +的CD11c-CD206 +细胞（Q4）。可以确认的BMDM激活状态通过增加细胞大小（ 图4A，右移FSC-A）和增加大量的表面抗原CD69，CD80，CD86或巨噬细胞（门:细胞CD11b + F4/80 +）， 如图4B所示 。产生细胞因子，基因表达和细胞信号转导通路激活M1或M2巨噬细胞中，可评估使用定量RT-PCR或免疫印迹。一显示在图5中，精氨酸酶1（Arg1）将和PPARγ水平增加M2中的巨噬细胞后，10 ng / ml的IL-4的刺激而增加在100 ng / ml的LPS刺激的巨噬细胞（M1）的IL-1β和TNFα产生，这是伴随着p65的活化（ 图6）。 <img src="/files/ftp_upload/50323/50323fig1.jpg" alt="图1" fo:content-width="5.5in"/> 图1。文中所述的隔离，形成和鼠标BMDMs刺激计划一步一步程序。简单地说，股骨和胫骨的骨骼收集6-8周的小鼠骨髓细胞用PBS中，用2％热灭活的胎牛血清补充冲出。氯化铵溶液裂解红血细胞后，细胞BMDM的生长培养基中培养7天，其次是成熟和纯度分析的细胞群。分析巨噬细胞宝<html>极化，将细胞用LPS或LPS刺激+IFNγ+ M1，或IL-4和/或IL-13的M2激活。偏振光的巨噬细胞的细胞形态的变化，表面标记上介绍，细胞因子的产生和细胞信号转导通路激活的基础上，可以进行评估。 <img src="/files/ftp_upload/50323/50323fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50323/50323fig2highres.jpg"/> 图2。流式细胞仪分析BMDM形成（A）BMDMs的第一门FSC和SSC除去杂物和共轭（B）成熟BMDMs被定义为细胞CD11b + F4/80 +亚（右上）的百分比显示的纯度母体门FSC / SSC。 s/ftp_upload/50323/50323fig3highres.jpg"/&gt; 图3。组织浸润的巨噬细胞， 巨噬细胞极化分析细胞CD11b + F4/80 +细胞被定义为M1巨噬细胞CD11b + F4/80 +的CD11c + CD206-细胞，，而M2巨噬细胞是细胞CD11b + F4/80 +的CD11c-CD206 +细胞。 <img src="/files/ftp_upload/50323/50323fig4.jpg" alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50323/50323fig4highres.jpg"/> 图4。激活后巨噬细胞活化流式细胞仪分析（A），巨噬细胞的大小，增加证明了移FSC-M1或M2巨噬细胞刺激相比，M0（未处理）的巨噬细胞在24小时后。（B）激活IL4或LPS刺激后流式细胞仪分析相关的表面标志。初期响应标记CD69进行评价，5或24小时的刺激后，CD80和CD86马克RS刺激后48小时进行了分析， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50323/50323fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50323/50323fig5.jpg" alt="图5"/> 图5。替代激活巨噬细胞，用10 ng / ml的IL-4刺激的巨噬细胞24小时，收集提取总RNA。 M2巨噬细胞，比未经处理的巨噬细胞，采用定量RT-PCR分析检测高架精氨酸酶1（ARG1）和PPARγ表达。 <img src="/files/ftp_upload/50323/50323fig6.jpg" alt="图6" fo:content-width="5.5in" fo:src="/files/ftp_upload/50323/50323fig6highres.jpg"/> 图6。古典激活巨噬细胞（A）提取总RNA，从巨噬细胞中与100纳克/ ML LPS为24小时，并用定量RT-PCR分析。在M1巨噬细胞促炎细胞因子，包括IL-1β和TNF-α的表达增加（B）NFκB通路的激活LPS诱导的确定使用抗体p65的磷酸化p65的蛋白印迹分析。

Watch this Scientific Journal Video about Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages at JoVE.com

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

本文介绍了一种自适应容易容易<em&gt;在体外</em&gt;模型，探讨巨噬细胞极化。在存在下GM-CSF/M-CSF，从骨髓中的造血干/祖细胞定向分化成单核细胞，由M1或M2的刺激。激活状态可以跟踪的细胞表面抗原，基因表达和细胞信号转导通路的变化。

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 ...

investigation-macrophage-polarization-using-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

66.2K 次观看.  Texas A&M University. 本文介绍了一种自适应容易容易在体外模型，探讨巨噬细胞极化。在存在下GM-CSF/M-CSF，从骨髓中的造血干/祖细胞定向分化成单核细胞，由M1或M2的刺激。激活状态可以跟踪的细胞表面抗原，基因表达和细胞信号转导通路的变化。

视频: Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

科研

免疫与感染

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

生物化学

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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.

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 ...

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 ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

<meta charset="utf8"></head><body>用于骨髓来源巨噬细胞分离、培养、极化和糖酵解表征的全面高效方案

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

用于骨髓来源巨噬细胞分离、培养、极化和糖酵解表征的全面高效方案

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

使用类似人类单细胞的THP-1白血病细胞系，将巨噬细胞分化和极化成M2样表型

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

使用骨髓源性巨噬细胞巨噬细胞极化的调查

摘要

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在小鼠体内的巨噬细胞的枯竭和重建

低氧环境下人巨噬细胞极化的蛋白质组学分析

一个

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

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

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

用于骨髓来源巨噬细胞分离、培养、极化和糖酵解表征的全面高效方案

用于骨髓来源巨噬细胞分离、培养、极化和糖酵解表征的全面高效方案

使用类似人类单细胞的THP-1白血病细胞系，将巨噬细胞分化和极化成M2样表型

使用骨髓源性巨噬细胞巨噬细胞极化的调查

摘要

探索更多视频

在小鼠体内的巨噬细胞的枯竭和重建

低氧环境下人巨噬细胞极化的蛋白质组学分析

一个

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

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

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

用于骨髓来源巨噬细胞分离、培养、极化和糖酵解表征的全面高效方案

用于骨髓来源巨噬细胞分离、培养、极化和糖酵解表征的全面高效方案

使用类似人类单细胞的THP-1白血病细胞系，将巨噬细胞分化和极化成M2样表型

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