这项工作得到了NIA R00 AG053412（KEF）的支持。

所有程序均按照《国家卫生研究院实验室动物护理和使用指南》的建议以及《涉及动物的生物医学研究国际指导原则》执行。该协议得到了北卡罗来纳大学夏洛特机构动物护理和使用委员会（协议#19-020）的批准。
1. 为文化做准备
注意:这些程序应在组织培养指定的生物安全柜中无菌条件下进行。有关媒体和解决方案，请参阅表 1。
<ol><li>通过在自密封灭菌袋中自动密封，对所有解剖工具进行消毒。</li>
	<li>准备多D-lysine的工作储备，用于处理12井文化菜肴。<ol>
<li>将聚D-lysine溶解在1x玻酸盐缓冲区中，浓度为100微克/mL（浓度为10倍）。在-20°C下存储大约1 mL的别名，直到需要为止。</li>
		<li>将 dPBS 中的 10 倍浓缩聚 D-lysine 稀释到 10μg/mL （1x）并过滤灭菌。</li>
		<li>在室温下以每口1x多D-lysine的0.5 mL处理12井培养板，至少1小时或过夜，供第二天使用。确保多D-lysine的体积足以完全覆盖文化区的底部。</li>
		<li>在使用之前，从培养板中取出多D-lysine，在无菌水中冲洗3倍，并保持无菌水，直到准备好板细胞。在电镀细胞之前，通过彻底的吸气去除所有液体的痕迹。</li>
	</ol>
</li>
	<li>准备神经元生长介质和 FACS 缓冲器。过滤消毒，并存储在4°C，直到使用。神经元生长介质可以保持约2周:FACS缓冲区可以保持约4周。</li>
</ol>2. 解剖胚胎海马
注:此程序可以在台面上执行，因为它需要使用立体显微镜来去除海马的脑膜和微分。坚持严格的不化菌技术，以尽量减少潜在的污染。
<ol><li>在CO2 室的胚胎日18日，对一只怀孕的C57BL/6J雌性小鼠实施安乐死。</li>
	<li>用 70% EtOH 喷洒腹部皮肤和毛皮，然后用组织钳捏皮肤，用手术剪刀做小切口。将内脏暴露在内脏和子宫内。</li>
	<li>用标准图案钳抓住子宫，抬出腔内，用细剪刀切断与子宫的连接。将带有胚胎的子宫转移到冰上放置的100毫米无菌塑料培养盘。</li>
	<li>使用细剪刀和细钳，小心切子宫，取出胚胎囊释放胚胎。将胚胎转移到一个新的100毫米无菌塑料培养皿中，其中含有放置在冰上的dPBS。</li>
	<li>用细剪刀斩首胚胎幼崽，并将头转移到新的100毫米无菌塑料培养皿中，其中含有放置在冰上的冬眠-E介质。</li>
	<li>要去除大脑，单手用一对细钳握住头部。使用弯曲的杜蒙#7钳在另一只手，插入在头骨底部的钳子尖端，捏骨头和过度的皮肤沿着中线前移动，而不会刺穿大脑。</li>
	<li>使用弯曲的钳子拉开皮肤和头骨，露出大脑。将大脑下弯曲的钳子从暴露的嗅球扫到小脑，将大脑从头骨中抬出。将大脑转移到一个新的100毫米无菌塑料培养皿中，其中含有放置在冰上的冬眠-E介质。每个大脑重复。 注:除非需要为实验特定目的分开，否则所有大脑都可以在单一培养皿中收集。</li>
	<li>在立体解剖显微镜下，一次用一个大脑和两对无菌的杜蒙#5钳子，捏掉嗅球，拉开脑膜。彻底切除脑膜是必要的，以避免培养污染的其他细胞，并允许进一步解剖。</li>
	<li>一旦脑膜被正确切除，皮层的上部侧向打开，这将暴露海马体。使用杜蒙#5钳，将海马从附着的皮层中捏开，并小心地将孤立的海马转移到冰上含有5mL冬眠-E介质的无菌15mL圆锥管。</li>
	<li>重复解剖两个大脑半球为每个胚胎小鼠幼崽，并结合所有海马到一个单一的15mL锥形管，除非需要单独用于实验特定目的。<ol>
<li>为了培养皮质神经元，皮层可能与皮层分离，处理与海马神经元相同。 注意:此时，协议可能会暂停。将大脑或解剖海马储存在冬眠-E介质中，辅以 B27 （2%）在4°C长达1个月，虽然延长延迟可能会损害可行的细胞的数量。</li>
	</ol>
</li>
</ol>3. 分离和培养海马神经元
注:所有程序应在组织培养指定的生物安全柜中无菌条件下进行。
<ol><li>为海马分离准备每个胚胎的0.5mL的木瓜分离溶液。所需的分离溶液量会因被解剖的胚胎大脑数量而异。 注意:对于皮质神经元培养，为每个胚胎准备1.0 mL的木瓜蛋白溶液。<ol>
<li>通过旋转约3分钟，每1 mL的冬眠E介质溶解20μL的木瓜因悬浮。</li>
		<li>帕潘溶解后，每1mL溶液中加入1μL的DNase I。在添加 DNase I 后不要旋转溶液，因为这样可以停用酶。</li>
	</ol>
</li>
	<li>离心机 15 mL 圆锥管包含海马组织 （步骤 2.10） 在 1，000 x g 5 分钟。删除超自然，并添加帕金解决方案。通过倒置几次来补充组织。在 37 °C 下孵育 30 分钟，每 10 分钟倒置一次组织。<ol>
<li>如果培养皮质神经元，将所有皮质转移到新的100毫米无菌培养皿中，小心地从盘子中吸气介质，用细剪刀或剃须刀刀片将肉末组织切碎。将木瓜素溶液加入培养皿中，并用木瓜溶液将皮质组织使用无菌转移移液器转移到 15 mL 锥形管中。在 37 °C 下孵化组织和酶溶液 30 分钟，每 10 分钟搅拌一次。</li>
	</ol>
</li>
	<li>在30分钟的孵化过程中，准备3个火抛光玻璃巴斯德移液器:1个完全打开，1个半开，1个季度打开。</li>
	<li>30分钟孵化后，离心机15mL圆锥管，内含消化脑组织，125 x g，10分钟。 取出酶溶液，代之以同等数量的新鲜冬眠 E 介质。</li>
	<li>使用完全打开的巴斯德移液器，将组织三次修剪10倍。让组织沉淀2分钟，然后将超自然细胞悬架转移到无菌的50mL圆锥管。将等量的冬眠 E 介质添加到组织中。</li>
	<li>使用半开巴氏杆菌移液器，三叶草组织10倍。让组织稳定2分钟，然后将超自然细胞悬架转移到与前一步相同的50mL圆锥管。将等量的冬眠 E 介质添加到组织中。</li>
	<li>使用四分之一打开巴斯德移液器，三叶草组织10倍。让组织稳定2分钟，然后再次将超自然细胞悬架转移到与之前步骤相同的50mL圆锥体。丢弃任何未分离的剩余组织。</li>
	<li>将含有超母体的50mL圆锥管从细胞悬架中分离出，在125 x g 下5分钟。如果尚未完成，则用无菌水清洗多 D-lysine 涂层板，并在离心过程中通过彻底吸入去除所有液体痕迹。</li>
	<li>离心后，丢弃超自然物，在神经元生长介质的5mL中补充细胞颗粒。使用尝试盘蓝色和血细胞仪数活细胞。<ol>
<li>如果培养皮质神经元，添加至少10mL的神经元生长介质，以减少细胞密度和更准确的计数。</li>
	</ol>
</li>
	<li>稀释具有神经元生长介质的细胞，最终电镀密度为每毫升 5 x10 5 个可行的细胞，并在 12 个井板的每口井中加入 1 毫升稀释细胞悬浮。将神经元保持在37°C，二氧化碳2为5%。 注:通常，海马从1个胚胎小鼠的大脑，即2海马，产生约1×106 个可行的细胞。这个数字仅用于实验规划目的，不应被视为每个培养细胞计数的替代品。</li>
	<li>在培养后的第二天改变生长介质，从每口井中去除一半的介质，即 0.5 mL，并在井边添加同等数量的新鲜介质，以避免破坏培养细胞。完成半媒体更改每周两次的文化寿命。</li>
</ol>4. 通过流动细胞测量评估MHCI表达
<ol><li>通过稀释干扰素β（IFN+）或神经元生长介质中等量的稀释剂来准备治疗。由于媒体更改将是半卷更改，使用 2 倍集中的 IFN® 准备治疗介质。即，用 100 U/mL IFN® 处理 12 口井板中的 3 口井，用 200 U/mL IFN® 或等量的 IFN® 稀释剂准备 1.5 mL，用于介质处理控制。</li>
	<li>从每口神经元井中取出 0.5 mL，并在每口井中加入 0.5 mL 的神经元生长介质（无论有没有 IFN®）。根据实验条件和信号优化，在正常生长条件下（37 °C，5% CO2）孵化6-72小时。</li>
	<li>治疗后，取出所有培养介质，在缺乏补充剂的冷神经巴介质（B27，L-谷氨酰胺，笔链）中轻轻洗一次。</li>
	<li>向每口井添加 0.5 mL 的非补充冷神经巴塞介质，其中含有 1μg/mL 的 Fc 块和 1 μg/mL 的荧光共聚抗 MHCI 抗体。在4°C下孵化，防止光线照射45分钟。</li>
	<li>取出含有抗体的介质，在冷 dPBS 中小心清洗一次。将 0.5 mL 无室温无酶细胞分离缓冲器添加到每个井中，并搅拌以驱离细胞。使用倒置组织培养显微镜观察分离。</li>
	<li>细胞从培养皿中分离后，向每口井添加 0.5 mL 的 FACS 缓冲器，将细胞三流化以分散团块，并将每口井的总体积转移到单独的 1.7 mL 微中微富格管中。</li>
	<li>离心机在 1，000 x g 5 分钟。取出超自然物，将细胞颗粒重新用100μL的FACS缓冲器，并将每个管子的总体积转移到96口井U底板的单独井中。</li>
	<li>在每口井中加入 100μL 的固定试剂。在室温下，在室温下进行多次三扰，以避免细胞聚集和孵育15分钟，不受光线照射。</li>
	<li>离心机在500 x g 5分钟。取出超自然物，并在 200 μl 的 FACS 缓冲区中重新发送。</li>
	<li>离心机在500 x g 5分钟。将含有荧光共聚抗NeuN抗体（1:100稀释）的渗透试剂中的超纳特去除并重新喷出。混合好，然后在室温下孵化，防止光线与摇动20分钟。</li>
	<li>孵化后，离心机在500 x g 下5分钟。在 100 μL 的 FACS 缓冲区中去除超自然并重新喷出。重复2次。</li>
	<li>最后洗涤后，在 FACS 缓冲区稀释的 100 μL 中稀释 2% 副甲醛的细胞。混合好以防止细胞团块。存储在4°C保护免受光，直到准备阅读流细胞仪。尽快阅读样品，在1周内。</li>
</ol>5. 量化和评估数据
<ol><li>测量每个荧光染料的补偿控制，并纠正任何光谱重叠。理想的补偿控制包括未染色的培养神经元，仅带有抗MHCI的污渍，仅带有抗NeuN。</li>
	<li>如果可能，为每个示例记录 100，000 个事件（至少 10，000 个事件），并保存为 FCS 文件。</li>
	<li>要分析数据，使用适当的分析软件，设置顺序门，如 图1A-C 所示，以选择NeuN阳性神经元。<ol>
<li>绘图SSC-A（日志）与FSC-A（线性）。在细胞群 （P1） 上绘制一个门，以消除分析中的细胞碎片。</li>
		<li>在 P1 蜂窝群中，绘图 FSC-H（线性）与 FSC-A（线性）。绘制单细胞群的门。</li>
		<li>在单个单元格群中，绘图 SSC-A（日志）与 NeuN（日志）。使用未染色或仅限 MHCI 的染色细胞绘制 NeuN 阳性人群的大门作为指南。</li>
		<li>绘制 MHCI 荧光的直方图，将细胞事件标准化为 y 轴上的模式。使用未染色或仅采用 NeuN 的彩色神经元作为指南，绘制水平门，以量化 MHCI 阳性的百分比神经元。</li>
		<li>出口 MHCI 的百分比细胞正数，以及 MHCI 在 NeuN 阳性人群中的荧光强度中位数 （MFI），用于统计评估和图形绘图。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

该协议描述了产前小鼠幼崽在胚胎第18天对原发性海马神经元的解剖和文化。使用由啮齿动物培养的原生神经元是现代神经生物学22中发展起来的最基本的方法之一。虽然不朽的细胞系可以模拟神经元的某些方面，它们作为肿瘤衍生细胞的性质，未能开发定义的轴突，以及持续的细胞分裂，令人怀疑它们是否忠实地重新概括了体内23个线粒体神经元的特性。原发性神经元的另一种替代方法是使用人类诱导的多能干细胞（HiPSC）。使用HiPSC的技术，特别是那些病人衍生的技术，近年来发展迅速。然而，与HiPSC合作仍有局限性，包括细胞系之间的变异性、缺乏功能成熟度以及表观遗传特征25的差异。虽然与原啮齿动物神经元的还原学模型合作也有局限性，但培养神经元在体内保留了神经元的后线性。此外，为小鼠提供的广泛的分子生物学工具和基因改造有利于在许多应用中使用原发神经元而不是HiPSC，小鼠研究可以很容易地转化为体内生物体中更为复杂的生物体，而不会失去实验基因系统。出于这些原因，许多研究人员使用原啮齿动物神经元来验证他们研究的关键方面，如果不是大部分的话。
对于某些检测，神经元可能直接分析后，从大脑前活体分离。 对于可能受特定实验条件或可能取决于多种细胞类型相互作用的成年小鼠的实验来说，这尤其可取:但是，有几个问题限制了可以进行的分析类型。从技术上讲，准备从成年小鼠的大脑中分离出单个细胞中止神经元是具有挑战性的，因为神经元是独特的相互连接和包围的骨髓26。组织三聚化的非酶方法在分离组织和导致细胞死亡方面效率低下，而酶制剂通常会裂开细胞表面抗原27。此外，虽然骨髓在胚胎小鼠中基本上不存在，但它约占成人大脑的20%，并可能损害可行的细胞分离和阻碍流细胞学分析28。许多已经开发的技术最终剥离他们的细胞溶胶神经元，并留下小，圆润的细胞体，主要由核29组成。虽然这在某些分析中是可以接受的，但这不适合量化细胞质或细胞外蛋白质表达。此外，减员细胞培养系统允许在较短的时间尺度上测试特定的机械问题，这比体内系统通常可能测试的时间要短。
本协议中还描述了用 IFN® 在药理学上刺激 MHCI 表达的方法，以及通过流动细胞学对细胞外 MHCI 表达的定量。IFN®的刺激是测试其他实验条件的有用阳性控制，但可以注意到，IFNγ和开尼酸也可以刺激MHCI表达在神经元9，30，而四恶英减少MHCI表达14。以往检测MHCI表达的方法依赖于原位杂交和免疫化学分析14、15、20、31。虽然基于 mRNA 的测定（如原位杂交和 qRT-PCR）可以确定空间空间定位、细胞类型特异性和基因转录水平，但这些测定无法评估蛋白质转化或传输到血浆膜。免疫化学和西式斑点分析可以确定蛋白质表达和潜在的细胞定位的差异，但可能难以准确量化。此外，许多MHCI抗体识别复合物的第三级结构，并且对构象变化高度敏感。因此渗透或变性条件导致MHCI免疫活性32的损失。此处介绍的方法用于 MHCI 的原位免疫维护，允许抗体在其原生构象中识别蛋白质，然后是固定和渗透方法。
稍作修改，此处描述的方法可用于培养其他神经元种群或评估其他感兴趣的细胞外蛋白质的表达。在此协议中注意到的是，为了培养皮质神经元，可以很容易地进行修改，但此处描述的方法也可用于培养其他神经元种群，如纹状神经元33。此外，虽然本协议规定了MHCI和NeuN的免疫染色，但其他细胞标记可以以类似的方式识别。一般来说，细胞外标记可以像MHCI一样处理，细胞内标记可以像NeuN一样处理。然而，应该指出，在细胞分离过程中，轴向投影与声瘤分离。由于此处定义的门控策略会筛选出细胞碎片，并侧重于神经元核标记物 NeuN，因此可能无法检测到完全在轴向投影中表达的蛋白质。
直到最近，神经元被认为表达MHCI只是为了回应损伤，感染，或体外细胞因子刺激，以从事细胞毒性CD8+T 细胞9。新的研究阐明了MHCI在13年发展过程中调节突触连接的另一个功能。此处描述的协议使用 IFN® 来刺激野生类型培养神经元的 MHCI 表达，但类似的方法可用于各种细胞刺激或基因改造，以测试特定假设。这种方法将使研究人员能够研究调节MHCI表达的分子机制，这将提高对MHCI在这两种不同的细胞功能中的二分法作用的理解。

中枢神经系统（CNS）曾经被认为是缺乏免疫监测，被称为"免疫特权1"。现在很清楚，这种特权并不等同于绝对缺乏免疫成分，而是一个专门的法规，其功能是限制与免疫病理学1相关的损害。事实上，CNS和免疫系统之间的沟通是一个持续的谈话，是健康的大脑发育和应对感染2，3所必需的。
主要组织相容性复杂I类（MHCI）分子是多态和多态的跨膜蛋白，最出名的功能是在感染4期间向CD8+T细胞呈现抗原肽。经典的MHCI复合物由跨膜α链和细胞外光链组成，称为+2微球蛋白。α链包含一个多态槽，结合抗原肽进行演示5。MHCI在细胞外膜上的正确表达需要内质视网膜的分子伴郎的协调作用，以确保α链和2微球蛋白的正确折叠，以及高亲和肽配体5的加载。只有组装完毕MHCI复合物，它们才能从内质视网膜出口到等离子膜6。当同源T细胞受体与肽加载的MHCI复合物接触时，CD8+T细胞通过释放含有穿孔素和颗粒酶的乳酸颗粒或通过目标细胞膜7上的结合Fas受体诱导凋亡来调节细胞杀伤。此外，CD8+ T细胞产生细胞因子，如伽马干扰素（IFNγ）和肿瘤坏死因子α（TNF®），它可以激活受感染细胞中的抗病毒机制，而不会产生细胞病变效应8，9。对于许多神经性病毒，CD8+ T细胞是清除来自CNS10，11，12感染所必需的。
以前人们认为神经元只有在损伤、病毒感染或体外细胞因子刺激的情况下才能表达MHCI。最近，研究确定了MHCI在突触重塑和可塑性13，14的神经元表达中的作用。虽然突触调控的精确机制尚不清楚，但数据表明，MHCI表达水平受突触活性15、16的调节。一种假设假定神经元会以先发制人地表达配对的免疫球蛋白状受体B（PirB），它使MHCI转瞬即失地结合到13、17。这种相互作用启动PirB的信号级联，反对参与突触重塑的路径，从而加强和稳定突触连接17，18，19。在没有神经元活动的情况下，MHCI表达减少14，MHCI的丧失导致突触消除有缺陷，突触回路20、21出现错位。
这里描述的检测结果改编自Chevalier等人9，它利用流动细胞学分析对MHCI在穆林海马神经元原生培养物上的细胞外蛋白表达进行定量评估。该协议说明了从胚胎小鼠幼崽身上微分海马组织的初始技术。然后，它详细说明了将组织酶和机械分离成单个细胞悬架的过程，以及维持体外培养物的方法。 因为它们不分裂，一旦它们处于培养中，神经元必须镀在适合其实验端点的盘子和密度中。接下来，它概述了用β干扰素（IFN+）诱导MHCI表达、MHCI和神经元核标记NeuN的免疫带状，以及通过流动细胞学分析细胞的步骤。最后，它描述了评估流动细胞测量数据以识别MHCI阳性神经元和量化MHCI表达水平的程序。本协议中还提到了可以进行的小调整，以便培养皮质神经元，而不是海马神经元。此协议可以很容易地修改，以测试特定的假设使用基因变异或药理治疗。

<table>
	<tbody>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td>Laboratory Supplies</td>
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			<td>100 mm sterile culture dish</td>
			<td>Fisher</td>
			<td>FB012924</td>
			<td>Tissue Culture Supplies</td>
		</tr>
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			<td>15 ml Centrifuge Tubes</td>
			<td>Genesee</td>
			<td>21-103</td>
			<td>Laboratory Supplies</td>
		</tr>
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			<td>50 ml centrifuge tube</td>
			<td>Genesee</td>
			<td>21-108</td>
			<td>Laboratory Supplies</td>
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			<td>500 mM EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td>FACS Buffer Reagent</td>
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			<td>70% Ethanol</td>
			<td>Fisher</td>
			<td>BP82031Gal</td>
			<td>Tissue Culture Supplies</td>
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			<td>96 well U-bottom plate</td>
			<td>Genesee</td>
			<td>25-221</td>
			<td>Flow Cytometry Supplies</td>
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			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>Neuron Culture Reagent</td>
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			<td>Borate Buffer</td>
			<td>Thermo Scientific</td>
			<td>28341</td>
			<td>Tissue Culture Supplies</td>
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			<td>Borosilicate Glass Pasteur Pipette</td>
			<td>Fisher</td>
			<td>13 678 20C</td>
			<td>Laboratory Supplies</td>
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			<td>Cell Dissociation Buffer</td>
			<td>Gibco</td>
			<td>13 151 014</td>
			<td>Flow Cytometry Supplies</td>
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			<td>DNase I</td>
			<td>Thermo</td>
			<td>90083</td>
			<td>Dissociation Solution Reagent</td>
		</tr>
		<tr>
			<td>dPBS</td>
			<td>Gibco</td>
			<td>14190-235</td>
			<td>Tissue Culture Supplies</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td>Dissection Tool</td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>Fine Science Tools</td>
			<td>91197-00</td>
			<td>Dissection Tool</td>
		</tr>
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			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>Tissue Culture Supplies</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>91113-10</td>
			<td>Dissection Tool</td>
		</tr>
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			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td>Dissection Tool</td>
		</tr>
		<tr>
			<td>Fix and Perm Cell Permeabilization Kit</td>
			<td>Invitrogen</td>
			<td>GAS003</td>
			<td>Flow Cytometry Supplies</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Neuron Culture Reagent</td>
		</tr>
		<tr>
			<td>Hibernate-E Medium</td>
			<td>Gibco</td>
			<td>A1247601</td>
			<td>Neuron Culture Reagent</td>
		</tr>
		<tr>
			<td>Instant Sealing Sterilization Pouches</td>
			<td>Fisher</td>
			<td>181254</td>
			<td>Tissue Culture Supplies</td>
		</tr>
		<tr>
			<td>Interferon Beta</td>
			<td>PBL Assay Science</td>
			<td>12405-1</td>
			<td>Flow Cytometry Supplies</td>
		</tr>
		<tr>
			<td>Milli-Mark Anti-NeuN-PE Antibody, clone A60</td>
			<td>MilliporeSigma</td>
			<td>FCMAB317PE</td>
			<td>Flow Cytometry Antibody</td>
		</tr>
		<tr>
			<td>Neurobasal Media</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Neuron Culture Reagent</td>
		</tr>
		<tr>
			<td>Pacific Blue anti-mouse H-2Kb Antibody</td>
			<td>BioLegend</td>
			<td>116513</td>
			<td>Flow Cytometry Antibody</td>
		</tr>
		<tr>
			<td>Papain Solution</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td>Dissociation Solution Reagent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Neuron Culture Reagent</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td>Neuron Culture Reagent</td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td>Dissection Tool</td>
		</tr>
		<tr>
			<td>Stereo dissection microscope</td>
			<td>Swift</td>
			<td>M29TZ-SM99CL-BTW1</td>
			<td>Dissection Tool</td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>91401-10</td>
			<td>Dissection Tool</td>
		</tr>
		<tr>
			<td>Transfer Pipets</td>
			<td>Corning</td>
			<td>357575</td>
			<td>Laboratory Supplies</td>
		</tr>
		<tr>
			<td>TruStain FcX (anti-mouse CD16/32) Antibody</td>
			<td>BioLegend</td>
			<td>101319</td>
			<td>Flow Cytometry Antibody</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154-100ML</td>
			<td>Tissue Culture Supplies</td>
		</tr>
	</tbody>
</table>

assessing the expression of major histocompatibility complex class i on primary murine hippocampal neurons by flow cytometry

越来越多的证据支持神经免疫相互作用影响神经系统功能在静态和病理条件下的假设。主要组织相容性复合I类（MHCI）的一个研究功能是向适应性免疫系统呈现细胞衍生的肽，特别是在对感染的反应中。最近，人们发现，MHCI分子在神经元的表达可以调节正常发育和神经紊乱期间突触连接中依赖活动的变化。这些功能对大脑健康的重要性支持了敏感检测的需求，这种检测很容易检测到神经元上的MHCI表达。在这里，我们描述了一种通过流动细胞学分析评估MHCI表达的穆林海马神经元原始培养方法。穆林海马体是在胚胎日18日从产前小鼠幼崽微分。组织使用酶和机械技术分离成单个细胞悬架，然后在无血清介质中培养，限制非神经细胞的生长。体外7天后，MHCI表达受到用β干扰素药理治疗培养细胞的刺激。MHCI分子被标记为原位，带有荧光标记的抗体，然后细胞被非酶分离成单个细胞悬架。为了确认神经元身份，细胞用副甲醛固定，渗透，并标记为荧光标记抗体，识别神经元核抗原NeuN。然后通过流动细胞学分析对神经元进行MHCI表达量定。神经元培养很容易被基因改造或药理干预来测试特定假设所操纵。稍作修改，这些方法可用于培养其他神经元种群或评估其他感兴趣的蛋白质的表达。

利用此处介绍的程序，海马组织在胚胎第18天从产前小鼠幼崽身上解剖出来。组织使用酶和机械方法分离成单个细胞悬架，然后在12个用多D-lysine预处理的井板中培养。体外7天后，细胞接受100U/mL的IFN®或介质治疗，只有72小时，这刺激了MHCI的表达。神经元在非酶分离成单细胞悬架之前，被污渍在MHCI原位。神经元被固定和渗透，然后在细胞内染色神经元核标记NeuN。样本通过流动细胞学进行评估，并使用相关软件分析数据。神经元通过对全部事件的连续门控来识别，以排除细胞碎片和双倍（图1A，B）。神经元由神经积极性（图1C）明确识别。NeuN+ 细胞通过在直方图上绘制细胞图，将细胞数量标准化到 y 轴上的模式，以及 x 轴上的 MHCI 荧光，进一步分析了 MHCI 的积极性。在正峰和负峰分化点绘制了 MHCI+ 闸门（图 1D）。从此，MHCI染色（图1E）和中位荧光强度（MFI: 图1F）计算。结果表明，IFN®治疗显著提高了MHCI细胞外污渍的神经元阳性百分比，以及MFI所示的表达水平。使用市售统计软件进行统计分析和图形表示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61436/61436fig1v2.jpg"> 图1:代表性门控策略和MHCI量化。 初级海马神经元仅接受100U/mL的IFN®或介质治疗。72小时后，神经元在细胞外与太平洋蓝共化MHCI（1μg/mL H2-Kb）染色，然后细胞内标记为PE-共聚NeuN（1:100稀释）。细胞荧光通过流动细胞学进行评估，并分析了数据。（A） 将事件总数绘制为 SSC-A（日志）与 FSC-A（线性），并将单元格 （P1） 封口以排除碎片。（B） 在P1种群中，细胞被绘制为FSC-H（线性）与FSC-A（线性），以门禁单细胞群。（C） 在单个细胞群中，细胞被绘制为 SSC-A（日志）与 NeuN-PE（日志）。NeuN+细胞被封闭以识别神经元。（D） 在神经元群中，细胞被绘制在直方图上，MHCI-PacBlu在x轴上，细胞数量正常化为y轴模式。绘制了水平门，以量化 MHCI 染色的神经元阳性的百分比。（E） 仅在介质和 IFN® 治疗神经元中对 NeuN +细胞的百分比 MHCI+进行量化。（F） 在介质（黑色）和IFN®治疗（红色）神经元中，对MHCI中位荧光强度（MFI）进行量化。统计学意义是通过未修损的 t 测试计算的。**，P<0.01。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61436/61436fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">神经元生长介质 （50 毫升）</td>
		</tr>
<tr>
<td>试剂</td>
			<td>最终浓度</td>
			<td>库存集中度</td>
			<td>对于 50 毫升</td>
		</tr>
<tr>
<td>B27 补充剂</td>
			<td>2%</td>
			<td>100%</td>
			<td>1.0 毫升</td>
		</tr>
<tr>
<td>L-谷氨酰胺</td>
			<td>2米</td>
			<td>200立方米</td>
			<td>0.5 毫升</td>
		</tr>
<tr>
<td>青霉素-链霉素</td>
			<td>100 U/毫升</td>
			<td>10，000 U/毫升</td>
			<td>0.5 毫升</td>
		</tr>
<tr>
<td>神经巴萨媒体</td>
			<td></td>
			<td></td>
			<td>48.0 毫升</td>
		</tr>
<tr>
<td colspan="4"></td>
		</tr>
<tr>
<td colspan="4">法克斯缓冲区 （500 毫升）</td>
		</tr>
<tr>
<td>试剂</td>
			<td>最终浓度</td>
			<td>库存集中度</td>
			<td>对于 500 毫升</td>
		</tr>
<tr>
<td>胎儿牛血清</td>
			<td>2%</td>
			<td>100%</td>
			<td>10 毫升</td>
		</tr>
<tr>
<td>埃塔</td>
			<td>1立方米</td>
			<td>500立方米</td>
			<td>1 毫升</td>
		</tr>
<tr>
<td>德普布</td>
			<td></td>
			<td></td>
			<td>489 毫升</td>
		</tr>
<tr>
<td colspan="4"></td>
		</tr>
<tr>
<td colspan="4">帕潘分离解决方案</td>
		</tr>
<tr>
<td>试剂</td>
			<td>最终浓度</td>
			<td>库存集中度</td>
			<td>对于 1 毫升</td>
		</tr>
<tr>
<td>帕潘暂停</td>
			<td>20 U/毫升</td>
			<td>1000 U/毫升</td>
			<td>0.020 毫升</td>
		</tr>
<tr>
<td>德纳塞一</td>
			<td>2.5 U/毫升</td>
			<td>2500 U/毫升</td>
			<td>0.001 毫升</td>
		</tr>
<tr>
<td>冬眠-E</td>
			<td></td>
			<td></td>
			<td>1.0 毫升</td>
		</tr>
<tr>
<td colspan="4">注意:所需的帕潘分离解决方案的体积会因被解剖的胚胎大脑数量而异。为海马神经元准备每个大脑0.5毫升，为皮质神经元准备每脑1.0毫升。</td>
		</tr>
<tr>
<td colspan="4">注意:准备分离解决方案，通过漩涡木瓜子暂停在冬眠-E约3分钟。帕潘彻底溶解后，加入 DNase I 。不要漩涡 Dnase I 。</td>
		</tr>
</tbody></table>表1:媒体和解决方案。

Watch this Scientific Journal Video about Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry at JoVE.com

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

该协议描述了一种从胚胎小鼠大脑中培养原发性海马神经元的方法。然后通过流动细胞学分析对培养神经元细胞外表面的主要组织相容性复杂I类的表达进行评估。

按流细胞测量评估主要穆林河马神经元的主要组织相容性综合类 I 的表达

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A ...

assessing-expression-major-histocompatibility-complex-class-i-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

3.3K 次观看.  University of North Carolina at Charlotte. 该协议描述了一种从胚胎小鼠大脑中培养原发性海马神经元的方法。然后通过流动细胞学分析对培养神经元细胞外表面的主要组织相容性复杂I类的表达进行评估。

视频: Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

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 ...

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 ...

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 ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

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 ...

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, ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous &#946;2m is not available. Meanwhile, it is indicated that mammalian &#946;2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m). Herein, methods to evaluate the feasibility of heterologous &#946;2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &#946;2-Microglobulin

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential &#946;2-microglobulin (&#946;2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous &#946;2m were investigated.

蝙蝠主要组织兼容性复杂类 I 的稳定性和结构与异质β2- 微球蛋白

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...

按流细胞测量评估主要穆林河马神经元的主要组织相容性综合类 I 的表达

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