这项研究是由澳大利亚中心艾滋病毒和肝炎病毒研究从华盛顿艾滋病研究中心大学（CFAR），根据奖号AI027757美国国立卫生研究院资助的程序，它支持（ACH 2）和2010年的发展拨款（CNIHR）资助通过下面的NIH研究所和研究中心（NIAID，NCI，镍氢电池，NIDA，NICHD，NHLBI，NIA）。 CSP是CNIHR和ACH 的赠款获得者。 SMC是澳大利亚（NHMRC）主要研究奖学金的国家健康和医学研究理事会的收件人。作者非常感谢由伯纳特机构收到了维多利亚经营性基础设施项目的支持这项工作的贡献。我们承认格扎Paukovic和伊娃ORLOWSKI - 奥利弗从AMREP流式细胞仪核心设施流式细胞仪培训和技术咨询协助。我们感谢安格斯摩根媒体教练和视频拍摄的组织。我们的感激杰西马森和讨伐异教徒阿卜杜勒阿齐兹K. Alzahrani在视频拍摄过程中的实验室协助。我们感谢大卫博士SIMAR在医学科学，新南威尔士大学，澳大利亚谁提供关键的方法建议，学校的努力。 CSP想感谢<a href="http://www.nice-consultants.com/">www.nice-consultants.com</a>用于图形磋商。 作者的贡献: CSP构思项目，设计并进行了实验，分析和解释数据，并且写了稿子。 JJA解释数据和撰写文章。 TRB写的稿子。 JMM解释的数据，提出关键知识的建议，并审查了原稿。 SMC解释的数据，做出关键的智力建议并审查了原稿。

注:艾滋病病毒感染者和艾滋病病毒感染者是从传染病股在阿尔弗雷德医院在墨尔本，澳大利亚招募，并从当地社区，分别为。从所有参与者获得知情同意书，研究批准了阿尔弗雷德医院研究伦理委员会。 在单核细胞和单核细胞亚群1.细胞的Glut1表面检测<ol><li>收集血液柠檬酸ACD-B的抗凝管并开始实验在生物安全柜收集1小时之内。 </li><li>加入100微升血液聚丙烯管。加2ml 1X的裂解液（见材料表 ）来管，而冰，移液轻轻混合。孵育在冰上15分钟。离心在220×g离心5分钟。 </li><li>倒出并通过加入大约2-4毫升洗涤溶液（在1×PBS中的0.5％BSA）并在220 xg离心离心5分钟洗涤两次。 </li><li>使用管道TTE要小心地取出尽可能多的洗涤液成为可能。在100微升洗涤液管放置在冰上重新挂起。 </li><li>确定具体的单核细胞亚群染色细胞与步骤1.4制得每100抗体微升细胞悬浮液的以下量:5微升抗CD3-PE，5微升抗-CD14-APC，5微升抗CD16-PECy7，5微升的Glut1 -FITC或的IgG2b-FITC（同型对照管）。 </li><li>在冰上放置在黑暗中30分钟。用洗涤液洗2次。 200-300微升在1X PBS在0.5％甲醛固定。 </li><li>以下激发和发射波长在24小时之内流式细胞仪能够检测4种颜色流分析:FITC（488，530），PE（488，575），PECy7（488，780），APC（633，660），10。 </li></ol> 2.葡萄糖摄取单核细胞<ol><li>收集在步骤1.1中的聚丙烯管移液器90微升的血液。添加10微升14.60μM2 NBDG工作液来90微升的血液（1.46毫米终浓度），并轻轻一抖混合。这是非常关键的覆盖管用铝箔限制2 NBDG曝光。 </li><li>孵育在37℃下在黑暗中15-30分钟，然后立即置于冰上。加4毫升1×FACS裂解解决管在冰冷。离心在4 220 XG °C为5分钟。 </li><li>通过加入4毫升洗涤溶液（0.5％在1×PBS中的BSA）洗涤一次。离心在4 220 XG °C为5分钟。倒出并在冰上的地方。 </li><li>染色细胞用抗体:5微升抗CD3-PE，5微升抗-CD14-APC和5μl抗CD16-PECy7。混合，并在冰上放置在黑暗中30分钟。 注:在此期间确保流式细胞仪已经准备好进行实时分析。获得以下几方面激发和发射波长范围内的单元格:2 NBDG（488，530），PE（488，575），PECy7（488，780），APC（633，660）。 </li><li> 4ml冰冷的洗涤缓冲液（在1×PBS中的0.5％BSA）添加到管中。离心220 XG洗一次在4 °C为5分钟。倒出并加入200-300微升冰旧的PBS，并保持在黑暗冰（用铝箔覆盖）。在流式细胞仪采用激发和发射波长设定在2.4步10分钟的流量进行分析。 </li></ol> 3.数据采集与分析注意:假设流式细胞仪和数据分析方面的知识。 <ol><li>用流式细胞仪能够至少4色分析的流，使用未染色和单独地染色样品设置的补偿。 注意:使用FITC标记的CD4和CD14单染色可用于的Glut1和2- NBDG补偿。 </li><li>建立和采集取样前标注相应的窗口。绘制围绕单核细胞群体的栅极，并获得每个样品100,000-300,000事件在中等速率。每个样品赔偿50,000个事件就足够了。 注:补偿之前，可以进行采样采集或在单细胞分析软件，以下的标准程序。 </li><li>出口和数据保存到一个适当的位置。打开的单细胞分析软件如的FlowJo或其他分析软件（ 参考图1）和拖放样品作为指定（ 参考图2）。 </li><li>双击打开文件（ 参考图3）。画一个圆门了一种基于单核细胞和图1A和补充图4所示侧向散射特性。双击单核细胞的人口。观察周围画了一个CD3箱-人口（ 参考图4）。 </li><li>双击CD3 -人口。观察单核细胞亚群上的"X"轴选择CD14-APC和CD16-PECy7上的"Y&#39;-轴，并相应标签（ 参考图5）。 </li><li>那里有没有明显阳性和阴性群，测量的Glut1在特定的单核细胞亚群表达或2-NBDG摄取。通过减去同种型和不2- NBDG背景（ 参考图6）确定的Glut1和2- NBDG的平均荧光强度（MFI）。 </li><li>其中，定义种群存在，则使用的IgG2b-FITC来设置的栅极，并确定百分比阳性细胞（ 图3）。 注:使用此方法来分析总CD14 +单核细胞。由于2-NBDG摄取通常是由在荧光移位标记强度数据最好由MFI和直方图表示。 </li></ol>

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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=Emerging+Role+and+Characterization+of+Immunometabolism:+Relevance+to+HIV+Pathogenesis,+Serious+Non-AIDS+Events,+and+a+Cure.">Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure.</a> J Immunol. 196 (11), 4437-4444 (2016).</li></ol>

The authors have nothing to disclose.

这里描述的协议细节的简单方法来检查在全血中的单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和荧光葡萄糖类似物的摄取。通过在全血评估-2- NBDG摄取，此技术允许类似于在体内条件。先前的研究中通过密度离心17日全血中分离的单核细胞检测6 NBDG摄取。然而，这项研究并没有审查的单核细胞亚群，并从全血中单核细胞的分离可以潜在地改变某些细胞表面分子19的表达。...

单核细胞是正在迅速动员到感染和炎症1的位点的人的先天免疫系统的重要组成部分。单核细胞的活化是限制由病原体产生急性损害的关键，也是中央对多种慢性疾病，包括动脉粥样硬化2，3癌症和HIV 4,5的发病机制。 静息和活化的单核细胞的代谢显着不同，以利用氧化代谢利用糖酵解代谢（ 即 ，葡萄糖发酵成乳酸）静息单核细胞和活化的单核6。单核细胞的活化诱导葡萄糖转运，可以增加葡萄糖摄取糖酵解代谢7的表达。单核细胞葡萄糖转运蛋白1（的Glut1）是激活期间上调一个这样的转运，其表达已显示导致生产v中促炎细胞因子的itro和在肥胖小鼠8的脂肪组织。由卡波西肉瘤相关疱疹病毒单核细胞系感染导致9的Glut1细胞上调，我们最近发现，慢性感染艾滋病毒期间的Glut1表达的单核细胞百分比增加是未经处理及联合抗逆转录病毒疗法治疗的感染10时在座。总而言之，这些研究表明，葡萄糖摄取和由单核细胞糖酵解代谢许多炎性疾病的重要方面。因此，一个简单的方法来测量稳态期间的单核细胞的Glut1表达和葡萄糖摄取和炎性疾病可能是使用了广泛的研究人员。 人单核细胞是异质的，正在由能够由细胞表面标记物CD14和CD16 11,12的差异表达进行检查三个不同的子集。经典单核细胞表达CD14的较高水平，但不表达CD16（CD14 + CD16 - ），中间的单核细胞表达CD14的高水平和CD16（CD14 + CD16 +）的中间电平，和非经典的单核细胞表达CD14的水平低和CD16高的水平（CD14 + CD16 +）。表达CD16的单核细胞被称为CD16 +单核细胞，这相比CD16 -单核细胞有炎性细胞因子的高表达和能力，更有效地呈递抗原13,14。单核细胞的大约10％与炎症15中观察到的更高的百分比的动态平衡期间表达CD16。单核细胞亚群与某些疾病状态相关联，并且可能是疾病和疾病进展16的有用的生物标志物。 我们的目标是确定可以测量通过在尽可能接近的phy条件人单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和葡萄糖摄取的方法siological条件越好。以前的研究测量的单核细胞葡萄糖转运蛋白的表达和葡萄糖摄取17,18，虽然这些检测方法可以比较生理条件19已经改变蛋白表达分离的单核细胞，并没有以前的研究调查了人单核细胞亚群。使用多参数流式细胞术，我们描述的方法来检查该荧光葡萄糖类似物2-NBDG按总单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和摄取（基于CD14和CD16的表达）全未经处理的血液内。

<table><tbody><tr><td>VACUETT Tube 9 ml ACD-B anticoagulant tubes</td><td>Greiner Bio-One GmbH</td><td>455094</td><td></td></tr><tr><td>5 ml sterile polypropylene tubes</td><td>BD Biosciences</td><td>352063</td><td></td></tr><tr><td>Albumin from Bovine Serum (BSA)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>16% formaldehyde solution</td><td>Electron Microscopy Science</td><td>15710</td><td></td></tr><tr><td>BD FACS lysing solution (10x)</td><td>BD Biosciences</td><td>349202</td><td>Dilute BD FACS lysing solution 1/10 with deionized water for working concentration (store for up to 1 week at 4 &amp;deg;C)</td></tr><tr><td>anti-CD3-PE</td><td>BD Biosciences</td><td>555340</td><td></td></tr><tr><td>anti CD14-APC</td><td>BD Biosciences</td><td>555399</td><td></td></tr><tr><td>anti-CD16-PECy7</td><td>BD Biosciences</td><td>557744</td><td></td></tr><tr><td>anti-Glut1-FITC</td><td>R &amp;amp; D Systems</td><td>FAB1418F</td><td></td></tr><tr><td>IgG2b-FITC</td><td>R &amp;amp; D Systems</td><td>IC0041F</td><td></td></tr><tr><td>2-NBDG</td><td>Life technologies</td><td>N13195</td><td>Suspend 5 mg of 2-NBDG into 1 ml of deionized water to make a 14.60 mM stock solution (keep for up to 6 months at 4 &amp;deg;C). To make the working 2-NBDG concentration, dilute stock 1/100 with 1x DPBS. Cover with foil. (store for up to 1 week at 4 &amp;deg;C)</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (1x)</td><td>Life technologies</td><td>14190-144</td><td>To make wash solution, add 0.5 g BSA per 100 ml DPBS (store for up to 2 weeks at 4 &amp;deg;C)</td></tr></tbody></table>

a simple flow cytometric method to measure glucose uptake and glucose transporter expression for monocyte subpopulations in whole blood

单核细胞是可通过与某些慢性炎性疾病相关的病原体和炎症被激活先天免疫细胞。单核细胞激活诱导效应职能和免受氧化糖酵解来代谢随之而来的转变，伴随增加葡萄糖转运蛋白表达。这增加了糖酵解代谢也为单核细胞经过培训免疫力，先天免疫记忆形式观察。虽然在体外单核细胞检查葡萄糖转运蛋白表达和葡萄糖摄取的协议进行了说明，没有已被多参数流式细胞仪在全血检测。我们描述了荧光葡萄糖类似物2-NBDG摄取全血中总单核细胞和经典（CD14 + CD16 - ）测定的多参数流式细胞协议，中间体（CD14 + CD16 +）和非经典（ CD14 + CD16 +）单核细胞亚群。该方法可用于检查稳态和炎症性疾病中总单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和葡萄糖摄取，并且可以容易地修改，以检查血液中的其它白细胞和白细胞亚群的葡萄糖摄取。

补偿必须单独荧光染料，以防止荧光溢出来执行。单核细胞首先通过选通基于前向和侧向散射富集。给出的图是从六个或更多个参与者对全血进行至少六个独立实验的代表如先前报道10 图1A示出了由CD3内选通由细胞散射和T细胞的排斥的单核细胞的最初选通-人口。然后单核细胞被选通为单独或与CD16以确定总单核细胞或单核细胞亚群的组合CD14的?...

Watch this Scientific Journal Video about A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood at JoVE.com

一种简单的流式细胞术方法测量全血中单核细胞亚群的葡萄糖摄取和葡萄糖转运蛋白表达免疫学和感染|视频

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

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16.4K 次观看.  Macfarlane Burnet Institute for Medical Research and Public Health. Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

视频: A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

科研

生物化学

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

免疫与感染

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

生物学

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, pro-inflammatory (i.e., M1-like) monocytes/macrophages display more reliance on anaerobic glycolysis and less reliance on mitochondrial oxidative phosphorylation, whereas anti-inflammatory (M2-like) macrophages display more reliance on glucose and fatty acid oxidation in the mitochondria. Here, we describe in-depth protocols for extracting macrophages from the two major monocyte/macrophage reservoirs in the body, the spleen and bone marrow, as well as injured tissues such as the heart following myocardial infarction.
Macrophages or monocytes are extracted by immunomagnetic sorting by using antibody-tagged microbeads, which easily bind to cells without compromising their phenotypes. The extracted cells are then cultured in 96-well plates, followed by extracellular flux analysis using a metabolic flux analyzer. Both glycolysis and mitochondrial oxidative phosphorylation can be measured simultaneously in small numbers of cells (as little as 2-3 &#215; 105 cells). This method can easily be performed in 1 day and produces reliable and repeatable results. Ultimately, these methods help to enhance our understanding of metabolic changes during immune and inflammatory responses to injury and disease, which could lead to the development of novel therapeutic targets for immunometabolic pathways.

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

Metabolic reprogramming is a hallmark of monocyte/macrophage activation and polarization between pro- and anti-inflammatory states. For example, ...

Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry

Source: Murinello, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54677">Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay.</a> J. Vis. Exp.&#160;(2016)
This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing neutrophils.

使用流式细胞术测量玻璃体内递送的荧光颗粒的小胶质细胞摄取

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

Source: Torchia, M. L. G., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58955">Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating strategy.

一种流式细胞术检测，用于鉴定外周血单核细胞中的免疫细胞亚群

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: This protocol has been tested ...

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

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细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

评估视网膜小胶质细胞吞噬功能

流式细胞仪定量测定GLUT4转位到质膜

全血流式细胞术分析人单核细胞亚群的表征

体外流动下单核细胞亚群的同时研究

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量

使用流式细胞术测量玻璃体内递送的荧光颗粒的小胶质细胞摄取

一种流式细胞术检测，用于鉴定外周血单核细胞中的免疫细胞亚群

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

摘要

探索更多视频

此视频中的章节

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

评估视网膜小胶质细胞吞噬功能

流式细胞仪定量测定GLUT4转位到质膜

全血流式细胞术分析人单核细胞亚群的表征

体外流动下单核细胞亚群的同时研究

分析脾和心脏巨噬细胞以及骨髓单核细胞中的 离体 代谢通量

使用流式细胞术测量玻璃体内递送的荧光颗粒的小胶质细胞摄取

一种流式细胞术检测，用于鉴定外周血单核细胞中的免疫细胞亚群

分析脾和心脏巨噬细胞以及骨髓单核细胞中的离体代谢通量