我们感谢丽贝卡西，MS和克里达纳里夫基金会动物加州大学欧文分校的核心基金为帮助他们在动物手术的技术人员。我们也感谢技术援助，手稿和视频拍摄的准备工作和动画加布里埃拉富内斯，乌莎Nekanti Denisse莫雷诺。这项研究得到了NINDS（RO1的NS43428 - 01到AJA），美国瘫痪项目（PPA - 32574 HXN），加州再生医学研究所（CIRM）干细胞培训奖T1 - 00008至HXN。韩国产业银行是由加州大学欧文分校（T32 NS007444）在分子和细胞神经科学培训计划的支持。

1。解离脊髓组织<ol><li>脊髓节段T8 - T10无伤或挫伤脊髓受伤（伤在T9）SD大鼠进行解剖和机械与罚款剪刀在室温下的HBSS分离，如前面所述1。组织分解之前，整个脊髓列前5分钟线细分T8 - T10提取存放在干冰。 </li><li>组织位被检索离心（1分钟的，为1000 rpm，室温）和酶2.5毫克胰蛋白酶和5毫克胶原酶5毫升二甲醚（贝科的修改鹰的媒体）20分钟，在37 °前trituration彗星（分离〜10倍在室温下）与巴斯德玻璃吸管（9英寸）。 </li><li>二甲醚+ 10％胎牛血清的10毫升加入到细胞，抑制酶的活动，然后通过一个40微米的细胞滤网过滤。快速旋转后，细胞沉淀悬浮于6毫升的HBSS OptiPrep梯度的解决方案如下所述重叠。 </li></ol> 2。创建OptiPrep渐变的解决方案<ol><li>摊薄OptiPrep构造OptiPrep 1:1稀释MOPS缓冲液（0.15 M氯化钠（NaCl），10毫米拖把，pH值7.4）。 </li><li>混合350，250，200，或150μL稀释1毫升（见表1）的最终体积的HBSS OptiPrep作了四个OptiPrep梯度的解决方案。 </li><li>四种解决方案是缓慢而小心地放置在一个15毫升的锥形管与底部至少淡化，最顶部（图1A）稀释层。 </li></ol> 3。从细胞中分离脂质/髓鞘碎片<ol><li> 6毫升的HBSS分离的脊髓细胞进行了仔细的分层的OptiPrep梯度解决方案上。 </li><li>离心管中的细胞和梯度解决方案（15分钟，1900 rpm或726 RCF 20 ° C）使用的Eppendorf离心机5810R斗摆动转子（A - 4 - 62），分成不同的层与脂质的电池解决方案/髓鞘碎片（前7毫升管），3层的神经元，与炎症细胞，神经胶质细胞，和红细胞颗粒。虽然大部分的炎症细胞，包括单核细胞，巨噬细胞，中性粒细胞粒，和淋巴细胞沉淀中发现，一些大型激活的巨噬细胞可以发现，在沉淀层以上。 </li><li>脂质/髓鞘碎片层（前7毫升）进行了仔细的吸气和删除。细胞，然后洗净，悬浮于2.5毫升的HBSS，用于免疫标记下面。 </li></ol> 4。特定的免疫细胞的免疫标记流式细胞仪<ol><li>颗粒细胞（500μL）收集脊髓准备5分钟，以溶解红细胞悬浮在0.85％的氯化铵（蒸馏水稀释）。 </li><li> 500μL的HBSS洗涤细胞，然后挡在正常兔或小鼠血清在室温30分钟。 </li><li>洗涤细胞，并在室温下孵育1小时抗体（FITC标记的兔抗中性粒细胞，精确的化学和科学;小鼠抗大鼠ED1 Alexa的488 Serotec;或小鼠抗大鼠CD3 Alexa的488）或同型IgG溶液稀释的HBSS（1:100稀释），如前面描述的1。 </li><li>洗涤细胞两次后的最后一步后每个重悬于300μL的HBSS上述步骤，然后。 </li></ol> 5。通过流式细胞仪检测细胞的定量评估<ol><li>一个流式细胞仪刀魂（流式细胞Dickinson公司）使用Cell Quest软件的流式细胞仪样本进行了分析。读取所有的样品，每个样品5000事件和首脑会议（DakoCytomation）完成数据分析。 </li><li>流式细胞仪门设置使用控制IgG抗体亚型标记的细胞或从没有受伤的控制动物的脊髓细胞正常化跨越时间点为基准值，前面描述1。积极标记细胞的平均值，表示为％（± SEM）的整个样本。 </li></ol> 6。代表性的成果： OptiPrep梯度清除髓鞘碎片和提高免疫细胞的流式细胞仪检测能力进行了评估量化脊髓中性粒细胞在1 DPI（图1B），中性粒细胞浸润以前1-3脊髓损伤后的高峰期。另外，相同的解剖脊髓无髓鞘碎片OptiPrep清除酶分离，并通过流式细胞仪检测中性粒细胞渗透的同时，同样，在评估。正如我们以前报道1，有OptiPrep梯度纯化方法相比，酶解单独隔离的样本中的事件的立场的转变，体现了完整的髓鞘碎片样本检测中性粒细胞的百分比只有一小部分（0.5％） OptiPrep purif检测中性粒细胞的百分比（5.1％）IED样品（图1B）。这些数据表明，在组织筹备工作的髓鞘碎片可以掩盖流式细胞仪的读数，并证明，髓鞘碎片的清除，增强了受伤的脊髓组织中的免疫细胞检测的灵敏度。 我们在建立OptiPrep梯度系统的细胞在脊髓损伤的检测灵敏度，超过期限从2小时到180 dpi的特点细胞浸润的变化，并建立一种新型的多相反应脊髓损伤后的炎症细胞，包括中性粒细胞，巨噬细胞/小胶质细胞和T细胞。 1-3以往的研究证实，中性粒细胞在伤后2小时首次进入线，并在1见顶DPI（图2和4） 。直到3 dpi的巨噬细胞/小胶质细胞， 另一方面 1,4,5，没有发现在脊髓损伤，并在7 DPI（图3＆4）初步见顶。令人惊讶的是，我们显示，巨噬细胞/小胶质细胞的第二次60 DPI达到顶峰，并保持180 DPI（图3＆4）升高的第一次。巨噬细胞/小胶质细胞，类似的T -细胞浸润高峰预计7 DPI 5-7;然而，流式细胞仪检测没有发现在第7 DPI在T细胞数量的任何变化，但检测到T细胞升高， 9 DPI（1.6％）（图4）。虽然T细胞的数量减少10 DPI，180 dpi的持久性T细胞的反应观察，当时4.4％的细胞被标记为CD3（图4）。 一起，这些数据表明一个SCI后的细胞炎症（图4）随时间变化的多相响应;细胞炎症的初期人的中性粒细胞1早高峰由DPI一个的ED1峰+巨噬细胞/小胶质细胞7 DPI和T其次细胞9 DPI，而在后期阶段，所有三种细胞与巨噬细胞/小胶质细胞的一个显着的第二个高峰期在60 dpi的人口上升后，14 DPI，并坚持通过180 dpi的，组成。这timecourse研究和流式细胞仪所产生的量化数据，先前已验证，从immunolabeled脊髓第1定量体视收集到的数据显示类似的结果。 <img src="/files/ftp_upload/2698/2698table1.jpg" alt="表1"/> 表1。 OptiPrep去除髓鞘碎片梯度准备。四个OptiPrep梯度的解决方案是使用的HBSS和稀释OptiPrep OptiPrep和拖把（1:1稀释）。 <img src="/files/ftp_upload/2698/2698fig1.jpg" alt="图1"/> 图1。OptiPrep梯度密度分开最受伤的脊髓组织/细胞的髓磷脂/碎片和提高免疫细胞的流式细胞仪评估。 （一）髓鞘/碎片分离后通过髓鞘/碎片层的愿望OptiPrep梯度离心分离脊髓组织细胞（神经元，神经胶质细胞和免疫细胞）。 （二）中性粒细胞在脊髓损伤，呈现在检测杂物清除后的中性粒细胞的敏感性增加（学生t检验，P = 0.0001）。所有流式细胞仪门，用未受伤的动物标记的细胞; N =每5组，平均± SEM 。每个样品5000事件被读取所有样品和积极标记细胞的平均值作为整个样本％（± SEM）的表达。 <img src="/files/ftp_upload/2698/2698fig2.jpg" alt="图2"/> 图2。中性粒细胞浸润流式细胞仪在受伤脊髓的检测。一个中度（200 KD）T9挫伤损伤后，中性粒细胞迅速开始（二）伤后2小时和调峰1 DPI（三）进入脊髓。所有流式细胞仪门是用未受伤的动物标记的细胞（A）。 <img src="/files/ftp_upload/2698/2698fig3.jpg" alt="图3"/> 图3的通过流式细胞仪在受伤脊髓的巨噬细胞/小胶质细胞浸润检测。一个适中（200 KD）在T9挫伤损伤后，ED1 +巨噬细胞/小胶质细胞数量增加损伤脊髓3 DPI（四）起，达到高峰在7 DPI（五）急性，在14 DPI（六）跌至较低水平前上升到60 DPI（G）的第二个高峰期，在脊髓损伤仍高达180 DPI（高）。 IgG抗体亚型7 DPI脊髓细胞的控制标签（二）表明最小的抗体标记的背景并没有从2小时抗体标记细胞受伤后（C）或没有受伤（一）动物的区别。所有流式细胞仪门是用未受伤的动物标记的细胞。 <img src="/files/ftp_upload/2698/2698table2.jpg" alt="表2"/> 表2。 timecourse实验动物样本 ，每个时间点（0小时为180 dpi），五禽戏在T9，一个中度（200 KD）挫伤损伤，通过流式细胞仪检测中性粒细胞，ED1 +巨噬细胞/小胶质细胞和CD3 + T细胞的数量进行了评估和脊髓组织。然而，并非所有的动物样本被成功恢复为中性粒细胞（4-5），ED1（3-5）和CD3（4-5）流式细胞仪分析。 <img src="/files/ftp_upload/2698/2698fig4.jpg" alt="图4"/> 图4。timecourse以下温和（200 KD）T9挫伤损伤脊髓细胞炎症。见顶敏锐地评估流式细胞仪，中性粒细胞的数量，ED1 +巨噬细胞/小胶质细胞，和CD3 + T细胞（1,7，和9 DPI，分别）和脊髓损伤的长期坚持。N = 3〜 5％组，平均± SEM。

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+inflammation+decreases+secondary+degeneration+and+functional+deficit+after+spinal+cord+injury.">Reducing inflammation decreases secondary degeneration and functional deficit after spinal cord injury.</a> Exp Neurol. 184, 456-463 (2003).</li><li>Stirling, D. P., Yong, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+inflammatory+response+after+murine+spinal+cord+injury+revealed+by+flow+cytometry.">Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry.</a> J Neurosci Res. 86, 1944-1958 (2008).</li></ol>

流式细胞仪的量化免疫细胞在中枢神经系统的使用是复杂的脂质/髓磷脂含量和碎片。除了 ​​与抗体结合的特异性干扰，髓鞘碎片可以有类似的免疫细胞的大小和肉芽属性，减少测量的灵敏度和准确性8。这里描述的新的细胞制备方法是有效去除髓鞘碎片，从而提高了检测脊髓损伤的细胞炎症的变化，流式细胞仪的灵敏度。举例来说，通过这种方法增强免疫细胞的检测的髓鞘碎片去除酶解alone.Critically相比，使用这种方法的新颖允许急性和慢性蜂窝炎症的第一表征脊髓损伤后，包括一个完整的中性粒细胞日久，巨噬细胞/小胶质细胞，T细胞为180 dpi，并确定了一个新颖的第二阶段，细胞炎症。这些重要的发现的一个neuroinflammation多相组成部分，可能是关键的设计和实施合理的治疗的治疗策略，包括脊髓损伤的细胞为基础的药物干预。 OptiPrep梯度系统已经使用单独的碎片从9血液中的细胞，或从脑细胞培养的目的10净化神经元;不过，我们已修改和先进的这种方法系统，单独从脊髓分离的细胞髓鞘碎片，并允许快速，更准确的定量流式细胞仪检测细胞炎症。可能这种方法用流式细胞仪，以提供在炎症研究中的重大进步后中枢神经系统受伤或病理条件下，作为大多数的研究都只能特征的形态组织学技术，是不是单元式专用或能不能始终提供真正的中枢神经系统的细胞炎症量化考核。此外，最近的一些研究已经试图量化流流式细胞仪使用新鲜分离的脊髓组织11,12或13 Percoll梯度系统分离细胞的免疫细胞在脊髓损伤，然而，这些研究证明或低细胞产生，或不敏感免疫细胞的检测。 相比之下，首次提供OptiPrep梯度细胞的制备方法，灵敏，可靠，流式细胞仪定量评估损伤的严重程度分级，并在延长timecourse高达180 dpi的细胞炎症的变化。流式细胞仪分析的灵敏度和可靠性，还取决于用于检测特定的免疫细胞类型的特异性抗体，流式细胞仪不允许形态标准，以进一步区分的细胞类型。中性粒细胞，巨噬细胞/小胶质细胞或T -告诉的特异性抗体已经彻底流式细胞仪进行测试，文化和细胞腹腔中性粒细胞和肺泡巨噬细胞在脊髓损伤组织1,2。连同OptiPrep梯度系统，用于流式细胞仪检测这些抗体作出贡献的一代进入SCI的微环境的动态提供了新的见解，并首次确定一个扩展的多相反应的细胞炎症反应的时间和定量分析细胞炎症。这种细胞在脊髓损伤后的多相反应可能是重要的调查目前在伤后的特定时间，特定的细胞类型的角色，或对特定的细胞类型，可加重受伤产生的治疗性干预。此外，这些研究结果可能有助于在一个适当的理由发展，为脊髓损伤的最佳药物或细胞为基础的的干预。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> OptiPrep </td><td> Fisher Scientific则</td><td> NC9182490 </td></tr><tr><td>的HBSS </td><td>西格玛</td><td> H1387 </td></tr><tr><td>兔抗大鼠中性粒细胞（FITC） </td><td>精确的化学与科学</td><td> AIFAD51140 </td></tr><tr><td>小鼠抗大鼠ED1（的Alexa Fluor 488） </td><td> ABD Serotec </td><td> MCA341A488 </td></tr><tr><td>小鼠抗大鼠CD3（FITC） </td><td> ABD Serotec </td><td> MCA772F </td></tr><tr><td>胰蛋白酶</td><td>西格玛</td><td> T9935 </td></tr><tr><td>胶原酶</td><td>西格玛</td><td> C5138 </td></tr><tr><td>二甲醚</td><td>西格玛</td><td> D1152 </td></tr><tr><td>鼠IgG1（的Alexa Fluor 488） </td><td> ABD Serotec </td><td> MCA1209A488 </td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30070.03 </td></tr><tr><td>兔血清</td><td>杰克逊ImmunoResearch </td><td> 011-000-120011-000-120 </td></tr><tr><td>小鼠血清</td><td>杰克逊ImmunoResearch </td><td> 015-000-120 </td></tr><tr><td>细胞过滤器</td><td>屋宇署猎鹰</td><td> 352340 </td></tr></tbody></table>

quantitative assessment of immune cells in the injured spinal cord tissue by flow cytometry:  a novel use for a cell purification method

免疫细胞在受伤的中枢神经系统（CNS）的使用形态或组织学技术检测并不总是提供真正的细胞炎症的定量分析。流式细胞仪是一种快速的替代方法来量化免疫细胞在脑损伤或脊髓组织。从历史上看，流式细胞仪已用于量化收集到的免疫细胞从血液中分离脾脏或胸腺，只有少数的研究试图量化流流式细胞仪使用新鲜分离的脊髓组织的免疫细胞在脊髓损伤。然而，分离脊髓组织集中髓鞘碎片可以被误认为是细胞，减少细胞计数流式细胞仪获得的可靠性。我们拥有先进的一个细胞的制备方法，使用OptiPrep梯度系统有效地分离脂质/髓鞘碎片从细胞，流式细胞仪在受伤脊髓细胞炎症反应灵敏，可靠的量化。正如我们在最近的一项研究（ 贝克＆阮等，脑2010年2月，133（PT 2）：。433-47），OptiPrep细胞制备增加了灵敏度，检测细胞在脊髓损伤炎症，与计数特定类型的细胞损伤的严重程度相关。更重要的是，这种方法的新颖使用提供的急性和慢性脊髓损伤后的细胞炎症的第一个表征，包括完成时间为多形核白细胞的过程（中性粒细胞，中性粒细胞），巨噬细胞/小胶质细胞，近一个时期的T细胞，从2小时伤后180天（DPI），确定一个令人惊讶的的小说细胞炎症的第二阶段。彻底表征细胞炎症，使用这种方法可能提供一个更好的neuroinflammation了解，在受伤的中枢神经系统和多相neuroinflammation揭示了一个重要的组成部分可能关键的设计和合理的治疗服务战略的实施，包括基于细胞和药理脊髓损伤的干预措施。

Watch this Scientific Journal Video about Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method at JoVE.com

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

在流式细胞仪受伤/病理中枢神经系统的细胞炎症的定量分析是复杂的脂质/髓鞘碎片，可以有类似的大小和肉芽细胞，减少敏感性/准确性。我们拥有先进的一个细胞的制备方法去除髓鞘碎片和改善由流式细胞仪检测在脊髓损伤细胞检测。

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

quantitative-assessment-immune-cells-injured-spinal-cord-tissue-flow

Research

JoVE Journal

Immunology and Infection

22.0K 次观看.  University of California. 在流式细胞仪受伤/病理中枢神经系统的细胞炎症的定量分析是复杂的脂质/髓鞘碎片，可以有类似的大小和肉芽细胞，减少敏感性/准确性。我们拥有先进的一个细胞的制备方法去除髓鞘碎片和改善由流式细胞仪检测在脊髓损伤细胞检测。

视频: Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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 ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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 ...

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 ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...