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/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/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 border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacutainers</td>
			<td style="width:125px;">BD Life Sciences</td>
			<td style="width:119px;"></td>
			<td>used for blood collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">WBC Fixative / RBC Lysis solution</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">00-5333-57</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD66b-APC</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">clone G10F5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45-APCeFluor780</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">clone 2D1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">s.aureus bioparticles</td>
			<td style="width:125px;">Life Technologies</td>
			<td style="width:119px;">A10010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dihydroethidium</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">D7008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N-ethylmalemide</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">4259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7AAD</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hematology Analyzer</td>
			<td style="width:125px;">Mindray</td>
			<td style="width:119px;">BC-3200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-channel pipet</td>
			<td style="width:125px;">Integra Biosciences</td>
			<td style="width:119px;">ViaFlo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bead Bath Incubator</td>
			<td style="width:125px;">LabArmour</td>
			<td style="width:119px;">BeadBath</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaging Flow Cytometer</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis FlowSight</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">INSPIRE Software</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis INSPIRE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IDEAS Software</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis IDEAS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">X-Pierce Piercable Plate Sealer</td>
			<td style="width:125px;">Excel Scientific, Inc.</td>
			<td style="width:119px;">X-Pierce</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dell Precision Workstation</td>
			<td style="width:125px;">Dell Computers</td>
			<td style="width:119px;">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/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/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

Research

JoVE Journal

Immunology and Infection

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

12.3K 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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

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, ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

整合影像流式细胞仪和转录组分析, 评估改变的内吞 CD1d 贩运

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...