Name: assessing retinal microglial phagocytic function in vivo using a flow cytometry-based assay
Uploaded: 2016-10-18T15:00:00
Description: Watch this Scientific Journal Video about Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay at JoVE.com

Salome Murinello is supported by American Diabetes Association grant #1-16-PDF-072. This work was supported by grants to Martin Friedlander from the National Institutes of Health (National Eye Institute EY11254 and EY22025) and the Lowy Medical Research Institute.

所有动物均按照由斯克里普斯研究所制定的道德准则处理。 1.注射材料的制备<ol><li>消毒一个33克的针头和注射器：在115οC.拆卸和高压灭菌器通过在无菌磷酸盐缓冲盐水（PBS）上升准备用于注射的针。 </li><li>解冻在室温下进行5荧光标记的粒子 - 10分钟。通过重构在无菌PBS 50毫克/毫升溶液中钙 /镁离子制备颗粒溶液注射。 注：已验证的吞噬检测真菌来源的AF488?...

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有在该方法中三个关键步骤：（1）荧光标记的粒子的玻璃体内注射液; （2）收集和准备视网膜组织;及（3）流式细胞仪分析。我们建议研究实行前履行我们在座的方法，玻璃体内注射。白化小鼠（ 例如 BALB / c）和有色溶液（ 例如，荧光标记的颗粒）可用于针的容易可视化和注入溶液。玻璃体内注射是具有挑战性的，如果不正确执行会导致偏见和不同的结果。与注射技术差相关的常?...

该方法的总的目标是要准确评估和体内小胶质细胞吞噬量化。小胶质细胞是中枢神经系统（CNS）的组织驻留的巨噬细胞。它们执行各种功能，以确保组织稳态的维持。这些包括免疫监视，神经营养因子的分泌，并为至关重要，吞噬作用1。小胶质细胞的吞噬作用是在诸如无关突触（突触修剪）和去除神经元凋亡2-4的吞噬大脑和视网膜的发育过程中的几个重要事件的关键。此外，损坏或凋亡细胞，细胞碎片和入侵微生物的小胶质细胞的吞噬作用已被证明是通过成年5维持CNS动态平衡是至关重要的。最后，小胶质细胞的吞噬作用已牵涉几种神经变性疾病，包括阿尔茨海默病和与年龄有关的发病黄斑变性，它已被认为有缺陷或不足的吞噬能力可能有助于淀粉样蛋白β（Aβ）斑块和玻璃疣，分别6,7的积聚。 小胶质细胞功能紧密可以通过微调节，特别是通过可溶性因子如肿瘤生长因子β或细胞 - 细胞相互作用。神经元组成型表达几个细胞表面配体，例如CD200和CX3CL1，而小胶质细胞只表达相应受体CD200R和CX3CR1。这些受体包含在它们的细胞内部分基于免疫受体酪氨酸的抑制基序（ITIMs）。这些抑制剂受体是用于防止小胶质细胞的过度刺激，从而可以向神经炎症的关键。因此，在正常生理条件下，神经元和小胶质细胞之间的细胞 - 细胞相互作用保持小胶质细胞处于静止状态。在组织损伤，然而，神经细胞可下调EXP这些配体的ression，除去小胶质细胞活化的抑制效果。小胶质细胞的功能（包括吞噬），因此紧密相连的微环境8。然而，到目前为止，还没有标准的测定法来研究小胶质细胞吞噬作用在生理环境或在完全复制其中枢神经系统微环境的一种方式。 几个实验已经开发来测量体外 ，其中primary胶质或小胶质细胞系是与靶细胞（ 例如 ，神经细胞凋亡），或荧光标记的珠培养的小胶质细胞的吞噬活性。那么目标摄取使用荧光显微成像或者流式细胞仪9-12评估。这些检测允许药理或遗传操纵可能会如何影响小胶质细胞的吞噬功能，虽然信息量大，无法完全复制体内环境的复杂的测试。审查小胶质细胞吞噬间接的方法在体内已经报道：这些是由被认为参与在吞噬作用（ 例如，CD68）分子的染色来实现，评估小胶质细胞和目标吞噬（ 例如 ，受损的神经元或突触元素）的物理接近度，或通过吞噬的免疫组化检测小胶质细胞内的目标（ 例如，Aβ）13-17。两项研究使用更直接的方法来评估在体内的小胶质细胞的吞噬功能。休斯和他的同事用成像技术来衡量通过颅内航线18条交付珠小胶质细胞摄取。塞拉等人开发了一种精制方法定量评估使用复杂的成像技术4凋亡细胞的小胶质细胞吞噬作用。然而，这些方法涉及用于组织制备，切片，成像和分析复杂的协议。我们在以前使用的流式细胞仪分析，以评估感光吞噬了通过在培养19视网膜色素上皮（RPE）细胞器段。这里，我们描述了协议来快速评估摄取荧光标记颗粒视网膜小胶质细胞在体内的小胶质细胞的吞噬作用的定量量度。 我们在这里描述的协议允许在三个关键步骤在不到6小时视网膜小胶质细胞吞噬的可靠和定量测量：（1）荧光标记的粒子的玻璃体内递送，（2）收获并制备视网膜组织的，和（3）流流式细胞仪分析。我们开发的方法是，以评估视网膜的小神经胶质细胞吞噬一个健壮的方法，它可以成功地用于测试各种不同的化合物或遗传操作如何改变在生理设置该键小胶质细胞的功能。由于中枢神经系统的一个专门区域，视网膜是研究小神经胶质细胞功能20的方便模式系统。虽然这种方法是在T开发他的眼睛，我们相信它可以为研究小神经胶质细胞吞噬功能全部神经学家有用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton&#160;syringe, 600 series</td>
			<td>Sigma</td>
			<td>26702</td>
			<td></td>
		</tr>
		<tr>
			<td>33 gauge, Small Hub RN NDL, 0.5 in, point style 4 - 12o</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>Z-23373</td>
			<td>Prepare immediately before injection</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>Need two</td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-10</td>
			<td>Curved</td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit &#8211; Postnatal Neurons</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-bottom Tube</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-bottom plate</td>
			<td>Falcon</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain Buffer (BSA)</td>
			<td>BD Biosciences</td>
			<td>554657</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-BV650 Antibody</td>
			<td>BioLegend</td>
			<td>101259</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly6C-APC-Cy7</td>
			<td>BioLegend</td>
			<td>128025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly6G-PE-Cy7</td>
			<td>BioLegend</td>
			<td>127617</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>BD Biosciences</td>
			<td>556463</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse CD16/32 Antibody</td>
			<td>BioLegend</td>
			<td>101301</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

在这里，我们描述了一种方法，使用流式细胞术分析（ 图2）迅速且可靠地量化在生理环境吞噬视网膜小胶质细胞的数量，该方法可适于测试上的吞噬能力的化合物和/或遗传操作的效果小胶质细胞（ 图3A，3B）。 （ - 1天产后10）或成年小鼠（ 图3C）也可在年轻使用。不同剂量的脂多糖（LPS）的腹膜内给药。 24小时后LPS攻击，这?...

Watch this Scientific Journal Video about Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay at JoVE.com

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

在组织的动态平衡和吞噬功能不足的维护工作在病理上被牵连的小胶质细胞的吞噬作用是至关重要的。然而， 在体内评估小胶质细胞的功能在技术上具有挑战性。我们已经开发出一种简单而坚固的技术用于精确监测和量化小胶质细胞的吞噬潜力在生理环境。

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, ...

Research

JoVE Journal

Immunology and Infection

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

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

科研

免疫与感染

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

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

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 ...

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

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 ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

使用IBA1和TMEM119进行小鼠脑微胶质密度、形态学和周围骨髓细胞渗透分析的免疫荧光染色

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...