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>

<ol>
	<li>Galea, I., Bechmann, I., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+immune+privilege+(not).">What is immune privilege (not).</a> Trends in Immunology. 28 (1), 12-18 (2007).</li><li>Morimoto, K., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Immune+System+in+the+Development+of+the+Central+Nervous+System.">Role of the Immune System in the Development of the Central Nervous System.</a> Frontiers in Neuroscience. 13, 916 (2019).</li><li>Klein, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+During+RNA+Viral+Infections.">Neuroinflammation During RNA Viral Infections.</a> Annual Review of Immunology. 37, 73-95 (2019).</li><li>Janeway, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T+cell+receptor+as+a+multicomponent+signalling+machine:+CD4/CD8+coreceptors+and+CD45+in+T+cell+activation.">The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation.</a> Annual Review of Immunology. 10, 645-674 (1992).</li><li>Peaper, D. R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+MHC+class+I+assembly+and+peptide+binding.">Regulation of MHC class I assembly and peptide binding.</a> Annual Review of Cell and Developmental Biology. 24, 343-368 (2008).</li><li>Donaldson, J. G., Williams, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Assembly+and+Trafficking+of+MHC+Class+I+Molecules.">Intracellular Assembly and Trafficking of MHC Class I Molecules.</a> Traffic. 10 (12), 1745-1752 (2009).</li><li>Charles, A., Janeway, J., Travers, P., Walport, M., Shlomchik, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell-mediated+cytotoxicity.">T cell-mediated cytotoxicity.</a> Immunobiology: The Immune System in Health and Disease. 5th edition. , (2001).</li><li>Guidotti, L. G., Chisari, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncytolytic+control+of+viral+infections+by+the+innate+and+adaptive+immune+response.">Noncytolytic control of viral infections by the innate and adaptive immune response.</a> Annual Review of Immunology. 19, 65-91 (2001).</li><li>Chevalier, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+are+MHC+class+I-dependent+targets+for+CD8+T+cells+upon+neurotropic+viral+infection.">Neurons are MHC class I-dependent targets for CD8 T cells upon neurotropic viral infection.</a> PLoS Pathogens. 7 (11), 1002393 (2011).</li><li>Shrestha, B., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CD8++T+Cells+in+Control+of+West+Nile+Virus+Infection.">Role of CD8+ T Cells in Control of West Nile Virus Infection.</a> Journal of Virology. 78 (15), 8312-8321 (2004).</li><li>Plaisted, W. C., Weinger, J. G., Walsh, C. M., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+mediated+suppression+of+neurotropic+coronavirus+replication+in+neural+precursor+cells.">T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells.</a> Virology. 449, 235-243 (2014).</li><li>Marten, N. W., Stohlman, S. A., Zhou, J., Bergmann, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+virus-specific+CD8++-T-cell+expansion+and+trafficking+following+central+nervous+system+infection.">Kinetics of virus-specific CD8+ -T-cell expansion and trafficking following central nervous system infection.</a> Journal of Virology. 77 (4), 2775-2778 (2003).</li><li>Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+I:+an+unexpected+role+in+neuronal+plasticity.">MHC class I: an unexpected role in neuronal plasticity.</a> Neuron. 64 (1), 40-45 (2009).</li><li>Corriveau, R. A., Huh, G. S., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+class+I+MHC+gene+expression+in+the+developing+and+mature+CNS+by+neural+activity.">Regulation of class I MHC gene expression in the developing and mature CNS by neural activity.</a> Neuron. 21 (3), 505-520 (1998).</li><li>Goddard, C. A., Butts, D. A., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CNS+synapses+by+neuronal+MHC+class+I.">Regulation of CNS synapses by neuronal MHC class I.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (16), 6828-6833 (2007).</li><li>Needleman, L. A., Liu, X. B., El-Sabeawy, F., Jones, E. G., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+I+molecules+are+present+both+pre-+and+postsynaptically+in+the+visual+cortex+during+postnatal+development+and+in+adulthood.">MHC class I molecules are present both pre- and postsynaptically in the visual cortex during postnatal development and in adulthood.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (39), 16999-17004 (2010).</li><li>Syken, J., Grandpre, T., Kanold, P. O., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PirB+restricts+ocular-dominance+plasticity+in+visual+cortex.">PirB restricts ocular-dominance plasticity in visual cortex.</a> Science. 313 (5794), 1795-1800 (2006).</li><li>Atwal, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PirB+is+a+functional+receptor+for+myelin+inhibitors+of+axonal+regeneration.">PirB is a functional receptor for myelin inhibitors of axonal regeneration.</a> Science. 322 (5903), 967-970 (2008).</li><li>Takai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+immunoglobulin-like+receptors+and+their+MHC+class+I+recognition.">Paired immunoglobulin-like receptors and their MHC class I recognition.</a> Immunology. 115 (4), 433-440 (2005).</li><li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+elimination+and+learning+rules+co-regulated+by+MHC+class+I+H2-Db.">Synapse elimination and learning rules co-regulated by MHC class I H2-Db.</a> Nature. 509 (7499), 195-200 (2014).</li><li>Ribic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+regulation+of+MHC+class+I+expression+in+the+developing+primary+visual+cortex+of+the+common+marmoset+monkey.">Activity-dependent regulation of MHC class I expression in the developing primary visual cortex of the common marmoset monkey.</a> Behavioral and Brain Functions. 7, 1 (2011).</li><li>Sahu, M. P., Nikkil&#228;, O., L&#229;gas, S., Kolehmainen, S., Castr&#233;n, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+primary+neurons+from+rat+hippocampus+and+cortex.">Culturing primary neurons from rat hippocampus and cortex.</a> Neuronal Signaling. 3 (2), (2019).</li><li>Gordon, J., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+overview+of+neuronal+cell+culture.">General overview of neuronal cell culture.</a> Methods in Molecular Biology. 1078, 1-8 (2013).</li><li>Zhang, X., Hu, D., Shang, Y., Qi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+induced+pluripotent+stem+cell+neuronal+models+to+study+neurodegenerative+diseases.">Using induced pluripotent stem cell neuronal models to study neurodegenerative diseases.</a> Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1866 (4), 165431 (2020).</li><li>Berry, B. J., Smith, A. S. T., Young, J. E., Mack, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+Current+Challenges+Associated+with+the+Use+of+Human+Induced+Pluripotent+Stem+Cells+in+Modeling+Neurodegenerative+Disease.">Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease.</a> Cells, Tissues, Organs. 205 (5-6), 331-349 (2018).</li><li>Ho, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Single-Cell+Transcriptomics+in+Adult+Rodent+Brain:+The+Medium+Spiny+Neuron+Transcriptome+Revisited.">A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited.</a> Frontiers in Cellular Neuroscience. 12, 159 (2018).</li><li>Panchision, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Flow+Cytometric+Analysis+of+Central+Nervous+System+Tissue+Reveals+Novel+Functional+Relationships+Among+Cells+Expressing+CD133,+CD15,+and+CD24.">Optimized Flow Cytometric Analysis of Central Nervous System Tissue Reveals Novel Functional Relationships Among Cells Expressing CD133, CD15, and CD24.</a> Stem Cells. 25 (6), 1560-1570 (2007).</li><li>Snaidero, N., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+at+a+glance.">Myelination at a glance.</a> Journal of Cell Science. 127 (14), 2999-3004 (2014).</li><li>Martin, D., Xu, J., Porretta, C., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurocytometry:+Flow+Cytometric+Sorting+of+Specific+Neuronal+Populations+from+Human+and+Rodent+Brain.">Neurocytometry: Flow Cytometric Sorting of Specific Neuronal Populations from Human and Rodent Brain.</a> ACS Chemical Neuroscience. 8 (2), 356-367 (2017).</li><li>Lv, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+MHC+Class+I+Expression+Is+Regulated+by+Activity+Driven+Calcium+Signaling.">Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling.</a> PLoS One. 10 (8), 0135223 (2015).</li><li>Barco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+facilitated+L-LTP+in+VP16-CREB+mice+reveals+that+BDNF+is+critical+for+the+maintenance+of+LTP+and+its+synaptic+capture.">Gene expression profiling of facilitated L-LTP in VP16-CREB mice reveals that BDNF is critical for the maintenance of LTP and its synaptic capture.</a> Neuron. 48 (1), 123-137 (2005).</li><li>Glynn, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHCI+negatively+regulates+synapse+density+during+the+establishment+of+cortical+connections.">MHCI negatively regulates synapse density during the establishment of cortical connections.</a> Nature Neuroscience. 14 (4), 442-451 (2011).</li><li>Facci, L., Skaper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Rodent+Cortical,+Hippocampal,+and+Striatal+Neurons.">Culture of Rodent Cortical, Hippocampal, and Striatal Neurons.</a> Methods Molecular Biology. 1727, 39-47 (2018).</li></ol>

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></tr><tr><td>100 mm sterile culture dish</td><td>Fisher</td><td>FB012924</td><td>Tissue Culture Supplies</td></tr><tr><td>15 ml Centrifuge Tubes</td><td>Genesee</td><td>21-103</td><td>Laboratory Supplies</td></tr><tr><td>50 ml centrifuge tube</td><td>Genesee</td><td>21-108</td><td>Laboratory Supplies</td></tr><tr><td>500 mM EDTA</td><td>Invitrogen</td><td>AM9260G</td><td>FACS Buffer Reagent</td></tr><tr><td>70% Ethanol</td><td>Fisher</td><td>BP82031Gal</td><td>Tissue Culture Supplies</td></tr><tr><td>96 well U-bottom plate</td><td>Genesee</td><td>25-221</td><td>Flow Cytometry Supplies</td></tr><tr><td>B27</td><td>Gibco</td><td>17504044</td><td>Neuron Culture Reagent</td></tr><tr><td>Borate Buffer</td><td>Thermo Scientific</td><td>28341</td><td>Tissue Culture Supplies</td></tr><tr><td>Borosilicate Glass Pasteur Pipette</td><td>Fisher</td><td>13 678 20C</td><td>Laboratory Supplies</td></tr><tr><td>Cell Dissociation Buffer</td><td>Gibco</td><td>13 151 014</td><td>Flow Cytometry Supplies</td></tr><tr><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><tr><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><tr><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://www-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

科研

免疫与感染

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

神经科学

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

神经细胞类型的流式细胞仪协议的表面和细胞内抗原分析

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

医学

使用流式细胞术进行小鼠脑组蛋白修饰定量

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

作者聚焦:基因表达调控研究的增强

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

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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通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰