The present study was funded in part by a Research Initiation Grant (RIG) from the University of North Texas to Dr. McFarlin. The authors did not receive direct funding for the completion of this study and report no conflict of interest.

注:此方法描述的所有血液采集程序均按照赫尔辛基宣言进行，批准UNT机构审查委员会（IRB）的人类受试者。所有受试者得到的采血，将其用于本发明的方法，以确保它们的正常体重明显健康，和无病的书面同意。 1.试剂来源及制品<ol><li>使用CD66b-APC（克隆＃G10F5; DF = 1:50）和CD45-APCeFluor780（克隆＃2D1; DF = 1:50），用于该测定。 注意:在此之前的研究中，CD45和CD66b的抗体滴度，以确定最佳稀释，从其它白细胞清楚地分辨粒（CD45 + / 66B +）（CD45 + / 66b-）11。在加入稀释的抗体的染色在管的最终稀释度为875。 </li><li>购买和解冻的股票S.金黄色葡萄球菌生物粒子标记盒pHrodo红色染料。暂停在1米的浓度克无菌PBS每毫升生物粒子的。等分试样稀释生物粒子和储存冷冻在-20℃直至在测定中使用。 </li><li>溶解二氢乙（DHE），它被转换为荧光的溴化乙锭中的氧自由基（ 即氧化突发）的存在下，在DMSO中，10微克/毫升的最终浓度。 </li><li>混合N-乙基马来酰亚胺（用于防止附加吞噬下列指定的测定温育持续时间）用无菌PBS以2步稀释200毫克/毫升和17.5毫克/毫升分别。在测定中，使用15mM的终浓度。当解冻N-乙基马来酰亚胺，使用37℃的热块，作为N-乙基马来酰亚胺不会留在溶液中。 </li><li>最终固定步骤后，用7AAD染色的核DNA。然后加入稀库存7AAD 1:10的用无菌PBS。 </li></ol> 2.采血<ol><li>要求受试者在实验室下面的O / N高速（&gt; 8小时），并abstenti到达从身体活动（&gt; 12小时）。 </li><li>清洗用酒精消毒片皮肤后，将无菌采血针到外围手臂静脉。 </li><li>采集血液注入真空管被商业化充满了肝素钠。 </li><li>收集后，取粘性绷带被摄对象的手臂。 </li><li>反转血液试管混合10次，然后放在一个摇杆直至分析。 </li></ol> 3.吞噬分析技术<ol><li>解冻葡萄球菌生物粒子（RT）下，DHE（RT），和N-乙基马来酰亚胺（37℃）。 </li><li>加入20升生物粒子金黄色葡萄球菌的4个人1.2毫升管在无菌罩工作时。 </li><li>添加40升DHE的含有该生物粒子每个管。 </li><li>轻轻拍打在板凳面管收集试剂的试管底部。 </li><li>添加100μL的混合全血到已填充有生物粒子和DHE每个管。 </li><li>擦拭管的内侧边缘与一棉签以除去任何污染的血液。 </li><li>混合血液和试剂与电子移液器设置为混合血液和试剂的血液加入后三个周期。 </li><li>将试管在冰桶和盖由轻保护。 </li><li>孵育测定管10，20和40分钟。先从40分钟管，以确保所有试管在同一时间完成。 </li><li>解冻N-乙基马来酰亚胺在37℃水浴珠。 </li><li>吸管15升N-乙基马来酰亚胺（上面1.4中描述）到每个测试管的，孵育30分钟。 </li><li>吸管10升CD66b-APC和10μlCD45-APCeFluor780稀释抗体（上述1.1中所述）的，孵育60分钟。 </li><li>吸取750升的WBC修复/ RBC裂解液到每个测试管，孵育60分钟。 </li><li>离心（10分钟，400×g离心）测定管，收集细胞沉淀。 </li><li>真空吸出WBC裂解/ RBC修复方案，留下的残余FLUID体积（100μL）中的细胞沉淀。 </li><li>吸管10μL的稀释7AAD溶液（上述1.5中所述）和50升的PBS到每个测试管。 </li><li>添加的校准珠（〜25L）向每个测试管，代替帽一滴上测定管，包裹管中的箔片，并在冰箱中的地方。 </li><li>装载样品管插入基于图像流式细胞仪和收集至少使用预先定义的参数3000粒事件:自动进样器，蓝色（488纳米; 60毫瓦），红（640纳米; 10​​0 NW），SSC（785纳米; 8.5毫瓦）的激光器（ 图1）。 </li></ol><img alt="图1" src="/files/ftp_upload/52201/52201fig1highres.jpg" /> 图1.采集方法和模板。被收购上的图像为基础的流式细胞仪（A）样品。在采集过程中，散点图的亮场宽高比与CD66b-APC生成从尖刺校准珠全光照分离粒克INSPIRE诉100.2.292.0软件（B）。 ，红色（640纳米，100 NW），SSC（785纳米; 8.5毫瓦）的激光器;使用自动进样器，蓝色（60毫瓦488纳米）分别获得每个样品最少5000粒。请注意，一些直方图是收购来监控激光设置和数据等方面收集时在座。所有这些直方图是可选的，只有在实验室标准操作程序要求他们作为质量控制的需要。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 4.样品采集与分析<ol><li>使用IDEAS软件的自动化软件补偿向导申请补偿矩阵原始图像文件（RIF），并创建补偿成像文件（CIF）。 </li><li>加载单独的CIF文件IDEAS软件，并生成以下地块以确定粒子集: </LI&gt; <li>建立初始门来识别被认为是在焦点使用直方图对明场梯度的RMS（ 图2A）的细胞。做出此判断，使用无刺激控制作为参考标准每个病人的样本。 </li><li>使用辅助图来从碎屑和双峰分离单细胞使用的明场纵横比（单元高度与宽度的比值）与明视场区域（ 图2B）的点积。做出此判断，使用无刺激控制作为参考标准每个病人的样本。 </li><li>一旦细胞干净人口已确定，建立的CD45与CD66b（ 图2C）的一个点图来正确识别粒（CD45 + / 66B +）。共创美好的细节强度S.的女儿图 （ 图3） 金黄色葡萄球菌 （x轴）与明亮的细节强度为氧化爆发（DHE，y轴）来识别激活的粒细胞的子集。收集BR在通道1和9飞行场图像，在通道2生物粒子，DHE在第4频道，7AAD在5频道，CD66b在11频道，和CD45在12频道。 </li></ol><img alt="图2" src="/files/ftp_upload/52201/52201fig2highres.jpg" /> 图2.分析模板，这个数字的系列被产生，以确定细胞分别在聚焦（A）中的曲线，单细胞（B），粒细胞（C）。附加地块被用来确定激活的粒与无活性的粒细胞的三个子集。获取的图像文件的所有分析使用IDEAS第6节软件完成。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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The authors have nothing to disclose.

本发明的方法代表了用于使用流式细胞1,3,4,12-14粒功能的评估现有方法细化。该测定过程的关键步骤往往会涉及到血液样品与所述生物粒子和DHE的适当的混合。不完全的混合会导致不准确的结果。而完全混合是非常关键的，混合方法应该是温柔的性质。有人建议，混合使用电子移液器具有混合功能，而不是一个涡流混合器来完成。在测定另一关键步骤是始终确保没有污染的血液测定管的上半部分。该残留的血液可以使用无菌棉签除去。完全除去是重要的，因为如果不这样做有可能污染最终测定制剂与未裂解的红细胞。 之前使用该方法适当补偿控制应加以控制对菌ectral重叠之间用于识别粒功能的各个方面的试剂。对于此方法，补偿控制涉及采集血液样本已被提出的40分钟测定孵育，然后用单个标记标记（ 即，大肠杆菌，DHE等）。标记后，单阳性事件的收集和使用的IDEAS分析软件的自动向导生成的补偿矩阵。至关重要的是，如果该测定的情况下，适当的补偿控制被完成，以确保适当的检测性能。 分析使用该功能查找和共定位向导来确定亮度细节强度分离人口的最好变量，还确定了如何氧化爆发和吞噬信号之间存在很多重叠完成。具体地，使用基于图像的细胞计数提供给分离激活的粒成三个亚群的能力。牛逼他的子集分类用吞噬作用与氧化爆发的亮度细节强度决定的。除了研究这些奇异细胞功能，细胞是在同一时间在相同的解剖学位置（共定位）显示出两个事件进行了鉴定。粒落在进入"高活性"的子集是该证明吞噬作用和氧化爆发之间是一致的共定位的唯一的表型。这种识别激活粒子集是在需要排除故障的最大面积。新用户花时间去了解IDEAS样品工作流程，并了解使用成像组件选通细胞群的机制是非常重要的。该用户可以寻求使其他修饰包括交替或额外测定的孵育时间的选择。当前的方法建议使用10-40分钟持续时间;然而，根据不同的实验模型，其中该方法是要使用时，它可能需要选择较长的测定持续时间。这样的修改将需要一个单独的基础上进行评估。 此外它被确定在测定孵育的持续时间，对三种活性子集的外观的显著效果。本报告中所描述的方法表示什么样的信息可以关于粒细胞功能3,4,13,15预先获得的扩展。其他实验室已经证明评估变化粒功能免疫力和抗病15-17全面评估的一部分的重要性。尽管该测定的电势也不是没有局限性。其中一个主要的限制是与高样品通量处理相关联的成本和时间的需求。当研究设计需要大量的在给定的一天的样品，这些可能难以处理。处理是通过使用电子吸管和掌柜精简，但这些往往是昂贵的，并且不一定可用在每一个实验室。 我们的研究领域集中在如何锻炼和饮食习惯影响免疫系统的健康和功能6,8-10,18-20研究。这些目标对多种人类健康领域显著实际影响。除了锻炼和营养作用的研究，在吞噬功能的监测是治疗效果的关键在这个手稿展示了吞噬的方法可能是在临床免疫学等领域非常有用。本试验是在第一许多与免疫学测定用潜力可重新发明通过取，可以是可以从图像的基于流式细胞仪的独特的成像信息的优点。

粒代表人体的先天免疫系统，它提供了抵御入侵抗原的第一行中的一个组成部分。以前的方法来评估粒细胞功能集中在吞噬能力或氧化突发使用独立的方法，从而使得难以确定如何集体粒已经改变1-4。在流动场进步术已导致在生产以高通量方式5能够高分辨率，多色细胞成像台式仪器的。成像与传统的流相结合的能力，流式细胞仪代表进步，提供给现有的流式细胞仪方法在创新和提取有关免疫系统的新信息所需要的技术平台。 在过去的十年里，我们的实验室，等等，已经敏锐地专注于各种营养和运动疗法patte效果RNS先天免疫功能6-9。在这个手稿证明该方法具有临床免疫学领域内的实际影响。本方法利用基于图像流式细胞仪的同时测量细菌粒子和氧化爆发吞噬的力量。使用这种方法，一个是能够​​分开使用由分析的基于图像的一部分提供的变量激活粒细胞。这些子集是评估对个体粒细胞图像后才辨认。进一步测定温育时间的影响三种激活子集10之间的过渡。因此，有理由认为使用多个孵育时间可以允许方法测试下面的特定的实验治疗粒细胞功能的变化。这份手稿的目的是通过使用基于图像的流式细胞仪同时测量与吞噬到oxidat演示评估粒细胞功能的方法香港专业教育学院爆裂。

<table><tbody><tr><td>Vacutainers</td><td>BD Life Sciences</td><td></td><td>used for blood collection</td></tr><tr><td>WBC Fixative / RBC Lysis solution</td><td>eBioscience</td><td>00-5333-57</td><td></td></tr><tr><td>CD66b-APC</td><td>eBioscience</td><td>clone G10F5</td><td></td></tr><tr><td>CD45-APCeFluor780</td><td>eBioscience</td><td>clone 2D1</td><td></td></tr><tr><td>&lt;i&gt;S. aureus&lt;/i&gt; bioparticles</td><td>Life Technologies</td><td>A10010</td><td></td></tr><tr><td>dihydroethidium</td><td>Sigma-Aldrich</td><td>D7008</td><td></td></tr><tr><td>N-ethylmalemide</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>7AAD</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Hematology Analyzer</td><td>Mindray</td><td>BC-3200</td><td></td></tr><tr><td>96-channel pipet</td><td>Integra Biosciences</td><td>ViaFlo</td><td></td></tr><tr><td>Bead Bath Incubator</td><td>LabArmour</td><td>BeadBath</td><td></td></tr><tr><td>Imaging Flow Cytometer</td><td>EMD Millipore</td><td>Amnis FlowSight</td><td></td></tr><tr><td>INSPIRE Software</td><td>EMD Millipore</td><td>Amnis INSPIRE</td><td></td></tr><tr><td>IDEAS Software</td><td>EMD Millipore</td><td>Amnis IDEAS</td><td></td></tr><tr><td>X-Pierce Piercable Plate Sealer</td><td>Excel Scientific, Inc.</td><td>X-Pierce</td><td></td></tr><tr><td>Dell Precision Workstation</td><td>Dell Computers</td><td>Various</td><td>Used for IDEAS analysis</td></tr></tbody></table>

image-based flow cytometry technique to evaluate changes in granulocyte function in vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

使用基于图像的流式细胞术使我们能够激活的粒的均匀种群分离成三种不同的激活子集（ 图3）。在该方法中，最有效的方法，以解决这三个子集的激活是通过绘制亮度细节强度为吞噬作用（ 金黄色葡萄球菌 ）与氧化爆发（DHE）（ 图3;图4）。另外，在IDEAS软件使用的共定位向导将允许同时吞噬作用和氧化爆发的存在，这是高度活化的粒细胞的标志符号的量化。 <img alt="图3" src="/files/ftp_upload/52201/52201fig3highres.jpg" /> 图3.识别激活粒细胞亚群。通过细胞表面标记（CD45 + / 66B +），粒细胞的识别后，女儿的情节与细节鲜艳产生INTENsity的吞噬与明亮细节强度氧化爆发。使用这种方法，不活动（紫色），低活性的百分比（红色的A），中等活性（蓝色，B）和高活性（黄色，C）的粒进行测定。这种门控技术也被用于评价测定孵育时间上的各种活性粒子集的相对丰度的影响。 "高活性"粒细胞比例最高的是目前继40分钟孵化。获取的图像文件的所有分析使用IDEAS第6节软件完成。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 除了演示活化粒细胞的三个不同的亚群的存在，证实了该测定的温育时间的影响的每个激活的粒子组的相对丰度。具体地，40分钟增量ubation导致的"高活性"粒细胞百分比最大。通过包括至少三个测定孵育持续时间有可能来确定如何在给定的临床治疗改变粒的瞬时激活状态。这是使用基于图像的流式细胞仪，以确定不同的活化的粒子集作为吞噬作用和氧化爆发的同时测量的函数的所述第一发布的方法。 <img alt="图4" src="/files/ftp_upload/52201/52201fig4highres.jpg" /> 细胞标记的 ，中等活性的（B）和低活性（C）的图4的代表图像。列为高活性（A）中的细胞的图像画廊示于该图中。 金黄色葡萄球菌生物粒子是CH03 ，氧化爆发是在CH04，7AAD的核心是CH05，边撒在CH06，CD66b是CH11和CD45是CH12。此外，被显示的吞噬（CH03）与氧化爆裂（CH04）的共定位合并图像。黄在合并图像区域表示的吞噬作用和氧化爆发都发生在同一时间同一解剖空间。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro at JoVE.com

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

image-based-flow-cytometry-technique-to-evaluate-changes-granulocyte

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

12.4K 次观看.  University of North Texas. This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

视频: Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

科研

免疫与感染

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

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within 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 ...

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 ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy 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, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

小鼠B细胞发育的流式细胞学表征

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

神经科学

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

生物学

Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry

Source: Do&#241;a, I. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62600">Basophil Activation Test for Allergy Diagnosis.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the test to evaluate basophil activation in the presence of a test allergen using flow cytometry.

使用流式细胞术在存在测试过敏原的情况下分析嗜碱性粒细胞活化

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sample preparation

	Collect ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

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

Source: Murinello, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.
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基于图像的流式细胞仪技术来评估变化粒细胞功能

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