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标记的粒子在这个协议中21-23使用。为了达到最佳的吸收，准备颗粒新鲜，并立即注射前。 注2:颗粒浓度可为每个特定实验进行优化。 </li></ol> 2.珠解决方案的玻璃体腔注射注:两个P需要eople执行注射，在某种程度上，使得执行注射可按住鼠标和保持的焦点上的眼球，而其他人传递加载的注射器和推动柱塞的人。 <ol><li>麻醉以100毫克/毫升氯胺酮和10mg / ml的甲苯噻嗪腹膜内注射的啮齿动物的剂量20微升/ 10克体重的。注射前，用脚趾捏来评估麻醉水平。 注:异氟醚对髓样细胞功能具有深刻的影响，因而，其在本测定法中使用，应避免24,25。 </li><li>负载针用0.5微升荧光标记的颗粒溶液。 </li><li>莱鼠标侧身手术显微镜（ 图1A-B）之下。使用软质材料， 如凝胶包，为鼠标的更好的定位。对于年轻小鼠中，眼睛还没有开放，轻轻创建一个fissu打开细45°直角钳的帮助下眼睑沿切口重新从眼睑最终将打开。 <ol><li>以优良的45°直角钳的帮助下仔细施加压力，周围的眼睑，使眼球轻微突出插槽。用两个手指就在耳朵上面并由其下颚握住头和并行皮肤轻轻拉伸到眼皮稍微保持眼睛从插座中。要小心，不要抓太靠近咽喉（ 图1B）。 </li></ol></li><li>穿刺眼球，将注射器的针头在角膜缘（其中角膜和巩膜连接）。这是在色素小鼠灰色圆圈可见。缩回针稍微驱逐玻璃体液中的一小体积，然后注入。在进行注射用一只手，并与其他的针应轻轻握住鼠标的人;第二个人应该慢慢推柱塞。 </li><li>慢慢地缩回注射器。敷眼滴保湿，保持眼部水分。&lt;/ LI&gt; <li>让啮齿动物在笼子里过隔热垫恢复，并继续监测动物。如果有幼崽时，请勿动物恢复到与其他动物的警戒，直到呼吸，并且能够自发运动的笼子。如果与成人的工作，不要啮齿动物恢复到与其他警报动物笼子里，直到它重新获得胸骨靠着。 </li></ol> 3.视网膜组织的收获注:从眼睛与荧光标记的颗粒注入视网膜组织应当收集作为流式细胞分析控制。虽然该试验可以使用单个视网膜中进行，以获得最佳性能，二视网膜应一起合并。 <ol><li>荧光标记的粒子的玻璃体内注射后收集视网膜组织3小时。 注意:当颗粒溶液注射后时间可以为每个特定实验进行优化，我们发现，在注射后3小时，粒子的摄取可以在大多数的层ö看出F中的视网膜（ 图1C-D）。 </li><li>通过颈椎脱位牺牲小鼠。 </li><li>通过对细45°角度的钳的帮助下proptose眼球眼睑轻轻按压收集眼球。放置眼球和拉后面的钳子。 </li><li>解剖解剖显微镜下视网膜。转移眼球到含有PBS中少量与钙 /镁离子培养皿。在培养皿的干燥区域，穿孔与超细镊子的末端的角膜缘的眼球。 </li><li>握住精45 度直角钳的帮助下，眼球和使用弹簧剪刀削减各地的角膜缘，直到角膜缘周围的大约一半被切断。 </li><li>握住精45 度直角钳眼球和带来眼球到PBS。随着第二对罚款45°直角钳撕裂的角膜和巩膜分开。该晶状体和视网膜会出来我ntact。 </li><li>分离晶状体和视网膜。收集视网膜和转移到含2毫升的PBS与钙 /镁离子一5.4毫升聚苯乙烯试管中。 </li></ol> 4.准备单细胞悬液<ol><li>准备使用具有小的修改，以制造商的说明一个神经元组织解离试剂盒单细胞悬浮液。简而言之，磨碎视网膜吹打向上和向下P1000的吸管和无晃动表演在37℃酶消化。 </li></ol> 5.染色单细胞悬浮液的流式细胞仪分析<ol><li>悬浮细胞在200μl染色缓冲液（Dulbecco氏磷酸盐缓冲盐水含有0.2％牛血清白蛋白（BSA）和0.09％叠氮化钠），并转移到一个U型底96孔板中。离心机在130×g下5分钟。 注:叠氮化钠对人体是有害和环境。使用适当的个人防护设备第二，根据当地法规丢弃垃圾。 </li><li>反转板在水槽上方，以弃上清。要阻止Fc受体，悬浮细胞在25微升含有抗CD16鼠/ CD32抗体的5微克/毫升，每孔染色缓冲。孵育在室温下5分钟。 </li><li>添加25微升含0.5微克/毫升的抗小鼠的Ly6C-APC-Cy7的的，抗小鼠Ly6G-PE-Cy7的0.5微克/毫升和2.5微克/毫升的抗小鼠的CD11b-AF650的染色缓冲液中。孵育在黑暗中在室温下15分钟。 </li><li>离心机在130×g下5分钟。反转板弃上清。通过在200μl的染色缓冲液中重悬浮细胞洗。 </li><li>离心机在130×g下5分钟。重悬在200μl含有0.5微克/毫升碘化丙啶（PI）的染色缓冲液中。转移至1.2ml微量管。 </li><li>洗井用另外100微升的染色缓冲液中含有0.5微克/毫升PI和池与前将200μl的1.2毫升微量管。得到的300微升染色的细胞的总体积。 </li></ol> 6.流式细胞仪分析<ol><li>使用传统的三个激光（紫色，蓝色和红色激光），避免PE，PerCP-经Cy5.5，BV510，并且由于显著溢出从非常明亮的荧光AF488粒子PI染料。使用的第四（黄色）激光来优化该试验中，因为它允许在PE标记的抗体和PI（在mCherry通道代替PerCP-经Cy5.5通道）的使用（ 图2）。 注意:如果一个黄色激光不可用，在另一个信道使用死细胞排除染料。 </li><li>门上的PI阴性细胞（PI-）排除死细胞（ 图2B）。 </li><li>不含死细胞，对细胞CD11b阳性细胞（细胞CD11b +）门之后，这将包括所有的骨髓细胞（ 图2C）。 </li><li>内的CD11b +人口，门上的Ly6C - / Ly6G -排除嗜中性粒细胞（细胞CD11b + / Ly6G +）和单核细胞（细胞CD11b + /的Ly6C +; 图2D）。门控小胶质细胞（ - / Ly6G -这里被定义为的CD11b + /的Ly6C为简单起见）后，两个明显的群体应该是可见的;一个负和一个正对荧光标记的粒子。吞噬细胞将已经采取了粒子，因此是AF488 +（ 图2E）。 注:的Ly6C积极或Ly6G阳性群体也可以利用分析这种染色方法来测量粒子的吸收。吞噬细胞CD11b +细胞的百分比与吞噬小胶质细胞的百分比（ 图2F）良好相关。研究人员不流式细胞经验，可以进行单独的CD11b染色更简单的分析，尽管这将包括吞噬嗜中性粒细胞和单核细胞，以及小胶质细胞。在此的CD11b + /的Ly6C - / Ly6G - &lt;/ SUP&gt;人口，这些细胞&gt; 99％的小胶质细胞通过用标记F4 / 80和CX3CR1染色来判断;这些标记可以添加，但并不在我们手中必要的。 </li></ol>

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The authors have nothing to disclose.

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见?...

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, ...

assessing-retinal-microglial-phagocytic-function-vivo-using-flow

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

9.5K 次观看.  The Scripps Research Institute. 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。 

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

科研

免疫与感染

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

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

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

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

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 ...

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).

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 ...

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 ...

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, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

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

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

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

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine 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

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

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

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

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

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

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

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

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

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

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

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

评估视网膜小胶质细胞吞噬功能

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