Name: analysis of immune cells in single sciatic nerves and dorsal root ganglion from a single mouse using flow cytometry
Uploaded: 2017-12-06T14:00:00.000+00:00
Duration: 12 min 34 s
Description: Watch this Scientific Journal Video about Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry at JoVE.com

这项研究得到了德意志 Forschungsgemeinschaft (DFG;SFB1118)。作者想感谢一周半跳 Erhardt 的大部分解剖和有用的传递这方面的知识, 并 Dr. 沃克 Eckstein 协助技术方面的流式细胞仪。

野生型 C57BL/6 小鼠 (雄性; 10-12 周) 被保存在标准的12小时光照/暗循环中, 并提供免费的标准膳食和水。所有动物实验都是按照当地动物保育和使用委员会的有关指导方针进行的, 并由德国卡尔斯鲁厄 (G216/10) 区域当局的当地动物保育和使用委员会批准。
1. 鼠标的灌注
<ol><li>牺牲鼠标 (C57BL/6;男性;10-12weeks) 使用 CO2或按照当地规定。 注: 使用 EDTA 涂敷的1.5 毫升管, 可进行逆行眶出血以获得控制白细胞。这种流血的方法只是在牺牲后立即有效。此外, 腹腔灌洗 (PCL) 可以获得控制巨噬细胞, 使用前加热 PBS 在37° c, 以避免冷凝集的血液, 这可能会妨碍灌注。</li>
	<li>用70% 伏/v 乙醇喷洒在腹部的皮毛, 用棉布纱布反复擦拭, 以消毒小鼠。使用钝剪刀, 切开腹部皮肤通过一个纵向切口。</li>
	<li>在胸腔内先用弯曲的钝剪刀将切口切开, 然后沿肋笼的整个长度延伸切口。在对侧做一个类似的切口, 直到胸骨可以被小心地抬走。</li>
	<li>用清晰的心和主要的血管, 插入一只蝴蝶23¾测量针, 连接到50毫升注射器充满冰冷的 PBS, 进入心脏的左室。</li>
	<li>用虹膜剪刀在心脏的右心房做一个切口, 尽可能大地创建一个插座, 而不会损害降主动脉, 然后轻轻地压低注射器, 使其通过大约30毫升的 PBS. 渗出液应该是清澈的。肝脏和/或肾脏的清除可以用来作为一个良好的灌注指标。</li>
</ol>2. 坐骨神经和脊神经节解剖
注意: 对于坐骨神经的解剖, 臀肌上方的皮肤被移除, 在伸展下肢的同时, 将老鼠置于腹侧向下。
<ol><li>双边解剖整个坐骨神经通过分离的肌肉组织从上背的大腿, 并沿着神经沿脊柱。切断神经, 它从脊柱的出口, 并立即在另一端的膝盖以上。 注意: 避免过度的神经处理, 如用钳子挤压或拉扯。</li>
	<li>使用镊子, 转移切除神经 (s), 约2厘米每神经, 1.5 毫升管含有1毫升冰冷 PBS 和储存在冰上。 注: 神经可以储存在冰上过夜。 注意: 对于背根解剖, 使用镊子和手术刀, 通过轻轻剥去残存的背部皮肤, 将皮肤从底层的软组织分离出来, 露出颈部、胸部和腰椎的区域。用钝的剪刀在头骨底部切开头部。</li>
	<li>在分离与脊柱前侧相连的内脏之前, 将肋骨与两侧的脊柱平行切开, 并将其靠近脊椎柱。切割股骨和删除脊柱的横向削减在水平的股骨。使用虹膜剪刀, 切断任何肌肉, 脂肪, 和其他软组织从脊柱。</li>
	<li>将脊柱与背侧朝上, 并打开它, 从尾端开始, 通过滑动剪刀的一个尖端到椎管内在三点位置和骨剪。在九点的位置重复步骤。 注意: 重要的是要准确地削减中心, 以避免损坏的背根。</li>
	<li>从侧面重复切割, 直到到达侧端, 脊髓完全暴露。使用解剖显微镜, 取出脊髓, 以揭露脊髓神经, 这可以用来定位在脊柱内的 DRGs。</li>
	<li>通过轻轻地拉动背部根部, 将背根拉入椎管内, 取出腰 DRGs, 然后用虹膜剪刀剪下神经和结缔组织, 去除 DRGs。 注意: 有可能限制收集到 L4-L6 背根, 这是助长坐骨神经。</li>
	<li>使用镊子, 转移 DRGs 到一个1.5 毫升管含有约1毫升冰冷 PBS, 并在冰上储存。 注: 神经可以储存在冰上过夜。</li>
</ol>3. 坐骨神经和脊神经节的消化
<ol><li>准备消化培养基 (DMEM, 10 毫米 HEPES, 5 毫克/毫升 BSA, 1.6 毫克/毫升胶原酶类型 4) 补充100µg/毫升 deoxyribonuclease, 并在冰上储存。 注: 每只老鼠所需的消化培养基量 (坐骨神经 + DRGs) 是1毫升。一旦准备好, 等分的消化介质可以储存在-20 ° c。</li>
	<li>将坐骨神经和 DRGs 从一只老鼠转移到单独的1.5 毫升管中, 其中含有500µL 消化介质。对于坐骨神经, 用弹簧剪刀将材料切成15-20 块, 然后在37° c 的摇动培养箱中孵育组织悬浮物, 20 分钟, 用柔和的搅动 (≤ 450 rpm)。</li>
	<li>反复研制细胞悬浮通过1000µL 吸管尖端机械离解任何不完全消化的组织。 注: 如果悬浮液难以滴定, 则用剪刀剪下吸管尖端以创建钝端。孵育组织悬浮在37° c 为20分钟以柔和的鼓动 (≤ 450 rpm)。</li>
	<li>重复研磨通过1000µL 吸管尖端和孵化为进一步10分钟在37° c 与温和的鼓动 (≤ 450 rpm)。</li>
	<li>准备的外地资产管制署缓冲 (PBS 补充 10% FCS 和1毫米 EDTA) 和储存在冰上。用 60 x 15 mm 的培养皿在冰上冲洗和浸泡一个网格大小为140µm 的不锈钢丝网。 注意: 应避免尼龙筛网过滤器, 因为它会导致有核细胞的产量显著下降。</li>
	<li>通过140µm 屏幕过滤器反复通过组织悬浮液, 因为它是在培养皿, 用钝钳, 在冰上举行。从培养皿中收集细胞悬浮液, 并将其转移到一个新的1.5 毫升的冰上储存。</li>
	<li>推动任何不完全消化的组织, 仍然在过滤通过重复研磨与1000µL 吸管尖端。冲洗屏幕过滤器两次与500µL 的外地资产管制系统缓冲区和收集结果细胞悬浮在培养皿结合与细胞悬浮从步骤3.6。 注: 如果使用的流式细胞仪对碎片敏感, 则步骤3.7 中的电池悬挂可通过70µm 滤网过滤;然而, 这可能导致有核细胞的产量的损失。</li>
	<li>离心的细胞悬浮在 300 x g 为5分钟在4° c。丢弃上清液, 在1毫升的重中进行细胞团的缓冲。重复这个洗涤步骤一次。</li>
	<li>重150-350 µL 的细胞团缓冲, 取决于所需的染色数量 (每染色面板150µL 的缓冲区)。</li>
</ol>4. 流式细胞术中荧光标记抗体对坐骨神经和背根的染色
<ol><li>将坐骨神经或背根细胞悬架的100µL 转移到一个96孔板上的 V 形井中。 注: 单染色细胞是必要的所有抗体, 因为他们将提供必要的控制, 以建立流式细胞仪关于光电倍增管 (PMT) 电压增益和补偿, 以及帮助区分特定的非特定绑定。由于来自坐骨神经和 DRGs 的有限的材料, 从 PCL (步骤 1.1-1.2) 的血白细胞和巨噬细胞可以作为单染色的控制, 其染色应与坐骨神经染色并行进行神经和 DRGs</li>
	<li>将剩余的细胞悬浮 (大约50µL 每样本) 从坐骨神经或根背根标本, 作为不控制。</li>
	<li>离心板在 400 x g 为 5 min 在4° c。通过把盘子翻转到垃圾箱上, 然后用手腕上的锋利的轻拍来丢弃上清, 然后轻轻地用纸巾擦干。</li>
	<li>对于表面染色, 重在20µL 中的每一个细胞球团都含有感兴趣的抗体 (CD45-A647, CD11b-PerCP/Cy5.5, 和 MHC II 类生物素) 和孵化, 保护免受光, 在冰上30分钟。 注: (1) 根据制造商的产品信息, 在使用稀释系列之前, 应确定最佳抗体稀释度;(2) 根据制造商的说明, 在这一步中, 通过 Fc 块孵化可以减少非特定的染色;和 (3) 同种控制染色通常是不需要使用的主要抗体直接与荧光和/或当抗原 (s) 的兴趣产生不同的人口。</li>
	<li>在每个井中加130µL, 并在4° c 下离心 400 x g 5 分钟。丢弃上清, 如前所述 (步骤 4.3), 并清洗细胞小球两次与150µL 的外地资产管制站缓冲区。 注: 如果使用游离主抗体或与生物素结合的主抗体, 则可在此时添加适当的二级试剂, 共轭于荧光, 并重复步骤 4.4-4.5。在本议定书的情况下, streptavidin-PE/Cy7 或亲和 PerCP 被用来检测 MHC 第二类与 CD45-A647/CD11b-PerCP/Cy5.5, 或结合 CD68-APC 和 F4/80-PE/Cy7, 分别。</li>
	<li>为固定, 在最后洗涤的步以后, 重细胞小球在100µL 4% 甲醛在 PBS (pH 7.4)。在冰上孵育板10分钟, 然后离心在 400 x g 5 分钟, 在4° c。丢弃上清, 如前所述 (步骤 4.3), 并清洗的细胞小球一次与150µL 的外地资产管制站缓冲区。 警告: 甲醛有毒;穿戴适当的保护例如手套和镜片,等。 注: 重150µL 中的细胞球团缓冲和存储保护免受光, 在 4 C 长达两天。如果不需要细胞内染色, 然后进行步骤4.11 后, 离心在 400 x g 5 分钟, 在4° c。</li>
	<li>对于细胞内染色, 重150µL 的细胞球团缓冲剂 + 0.5% 洗涤剂, 并在室温下孵育, 保护免受光15分钟. 离心板在 400 x g 为 5 min 在4° c。丢弃上清, 如前所述 (步骤 4.3) 和洗涤的细胞颗粒一次与150µL 的外地资产管制的缓冲 + 0.5% 洗涤剂。重20µL 的细胞球团缓冲剂 + 0.5% 洗涤剂含有的抗体的兴趣 (CD68-APC, CD206-PE) 和孵化, 保护免受光, 在室温下为30分钟。</li>
	<li>增加130µL 的外地资产管制的缓冲区 + 0.5% 洗涤剂每井和离心板在 400 x g 5 分钟, 在4° c。丢弃上清, 如前所述 (步骤 4.3) 和洗涤的细胞小球两次与150µL 的外地资产管制的缓冲区 + 0.5% 洗涤剂。 注: 如果使用游离主抗体或与生物素结合的主抗体, 则可在此时添加适当的二级试剂, 共轭至荧光, 并重复步骤 4.9-4.10。</li>
	<li>重150µL 的细胞球团缓冲 + 0.5% 洗涤剂和商店, 保护免受光, 在4° c 最多两天。</li>
	<li>重的细胞小球与150µL 的缓冲区 + 0.5% 洗涤剂补充5µg/毫升 DAPI 和孵化5分钟室温下, 保护免受光。离心板在 400 x g 为 5 min 在4° c。丢弃前述的上清液 (步骤 4.3)。</li>
	<li>重150µL PBS 中的细胞小球, 并转移到一个适当标记的外地资产管制管。储存在室温下, 免受光照, 直到准备好分析。 注意: 在 PBS 悬浮后, 细胞应在数小时内进行分析, 因为 DAPI 会慢慢脱离 DNA, 导致信号丢失。</li>
</ol>5. 流式细胞仪的设置和样品的运行
<ol><li>确保流式细胞仪配备适当的激光器和/或过滤器集, 以测量用于染色荧光的电池。这可以在系统的默认的细胞仪配置下确定。在本协议的情况下, 使用流式细胞仪进行分析, 配备三激光器 (405 nm、488 nm 和 633 nm), 允许检测≤5荧光。</li>
	<li>选择适当的渠道, 以检测荧光的利益。所有的荧光信号最好的检测与对数放大。</li>
	<li>为样本选择适当的流速。这个参量可能变化取决于使用的系统, 并且应该经验主义地被确定。在本协议的情况下, 一个流动率的 MED 到 HI (35-60 µL/分钟) 被用来防止堵塞的针。</li>
	<li>选择要在每个示例中收集的事件/单元格的总数。最小事件数应为100万。</li>
	<li>在全局工作表下创建散点图-a 与 FSC-a, 以及每个荧光的兴趣与 FCS a。创建一个统计视图, 它将显示所选荧光的事件数和平均荧光强度。</li>
	<li>设置 FSC 的电压-a 和 SSC-一个通道, 使最小的单元格符合散点图的左下10%。从坐骨神经或 DRGs 的不控制中确定背景荧光和最小样品荧光. 根据 DAPI 染色控制的散射图确定父 (P1) 门。 注意: 这不是一个集合门, 部分原因是, DAPI + 种群的异质性。</li>
	<li>使用 FSC 分散图为每个荧光的利益, 调整电压, 使不控制的细胞在其各自的散射地块的下四分之一。然后在这个区域之上创建门, 以定义那些对每个标记/荧光都是阳性的细胞。通过单染色控制 (步骤 4.1) 的运行, 确定了浇口的位置。 注: 重叠发射谱的补偿可以在这一点上进行, 或者追溯到适当的分析软件。</li>
	<li>一旦流式细胞仪已经建立, 如所述, 样品可以运行。</li>
	<li>在运行后, 剩余的样品材料可以被丢弃, 并可以导出 FCS 文件, 以便进一步分析。</li>
</ol>

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作者在这项研究中没有利益冲突。

坐骨神经含有很大比例的脂质, 如胆固醇, 由于髓鞘周围的神经轴突的内容。由于油脂的性质随温度变化, 在不同的温度下可以得到不同的结果。为了保证细胞的保存, 消化后的所有步骤都在冰上进行。为了使结果重现性起见, 建议保持一致性, 但在室温下执行步骤3.6 和3.7 可以提高产量。如果3.8 步的神经没有被充分消化, 小块神经将在网格上可见。在不充分消化的情况下, 在网格上保留的材料可以通...

从循环中进入三七总皂苷的免疫细胞, 如 CD45 和 CD11b 的表达, 有助于维持神经的完整性, 并在再生和变性方面发挥作用1。巨噬细胞 (由它们的 CD68 在老鼠中的表达) 可以偏向于炎症表型, 表达更多的 MHC 类 II 和 CD86 在他们的表面 (M1), 或对抗炎表型, 表达更多的胞内 CD206 (M2)2.巨噬细胞表型的倾斜是通过 Akt 信号3调节的动态过程, 反映了巨噬细胞在防御病原体 (M1) 和组织再生中的作用 (M2) 的不同任务。损伤的神经的再生首先要求吞噬髓鞘残骸由巨噬细胞在神经4,5和抗炎性 (CD68+CD206+) M2 巨噬细胞被证明促进轴突生长在6。减少招募或巨噬细胞的总皂苷, 或受损能力的吞噬可能导致受损的再生和维持神经完整性。炎症 M1 巨噬细胞, 表达 MHC 类 II, 是较少的能力吞噬比 M2 巨噬细胞, 和神经元炎症牵连的发病机制的几个神经退行性疾病7。
由于损伤引起的神经居民免疫系统的变化可能是定量的, 表现在失去 CD45 的+白细胞 (或增加渗透的 CD45+白细胞的情况下炎症), 或定性, 如巨噬细胞表型从 M2 到 M1 表型的变化。通过使用冰冻切片或石蜡嵌入材料8, 对三七总皂苷的免疫细胞进行了传统的分析。如果需要确定感兴趣的细胞的定位。然而, 定量使用如果经常依靠计数少量细胞在一个狭窄的部分组织, 使定量不可靠和易受选择偏倚。为了识别特定的免疫细胞子集, 同时检测细胞外和/或细胞内标记是必需的, 而巨噬细胞表型的确定至少需要两个标记, 特别是 CD206 和 MHC类 II。由于大多数常用的显微镜都被限制在至少两色的通道, 如荧光素异硫氰酸酯 (FITC) 和藻 (PE), 对免疫细胞的特定子集的表征, 如果可以是限制性和不完整的, 需要需要有多个幻灯片, 从相同的兴趣领域, 这是染色和分析并行。因此这费时的方面不必要借自己对大样品集合的分析。此外, 由于大部分的兴趣标记都是胞外的, 所以在组织中的检测, 或者被植入石蜡或 cryoconserved, 由于细胞膜完整性的破坏和表位的掩蔽, 以及失去由使用溶剂引起的抗原, 如丙酮和甲醇9。
相比之式细胞仪 , 它测量单细胞的光学和荧光特性的悬浮 , 因为他们通过一束光 , 提供了一个更实用和全面的方法来分析细胞的数量。流式细胞术, 而不是制作一个数字图像的组织, 提供了一个自动量化的设置参数, 其中包括一个细胞的相对大小和反射指数, 称为前散射 (FSC), 粒度/内部复杂度或侧散射 (SSC) 和相对荧光强度, 提供该细胞已被标记为适当的荧光, 如共轭抗体。典型的流式细胞仪由两个空冷激光器组成;氩激光在 488 nm 处产生蓝光, 氦氖激光器在 633 nm 产生光。这一组合允许检测和测量, 同时, 至少五目标, 无论是在表面上或在细胞内室。更先进的流 cytometers 可以由多个激光器组成, 它允许一次检测多达八种不同的荧光, 这就使得所选荧光的峰值发射波长不会有明显的重叠。
通过流式细胞仪分析, 首先要酶消化, 一般用胶原酶, 产生单细胞悬浮。小鼠坐骨神经的分析以前是困难的, 因为少量的组织从每只老鼠获得。此外, 高脂肪含量的髓鞘周围的轴突阻碍细胞的恢复和产生大量的碎片。本文对坐骨神经的制备和消化的方法进行了改编, 从发夹的雪旺细胞隔离协议的et al.7, 目的是从单个小鼠的神经中分离出足够的细胞进行流式细胞仪分析, 以减少小鼠之间的变异。在原始组织文摘中使用 DAPI 来识别单个细胞, 绕过需要去除轴突碎片, 这通常会导致细胞丢失。清洗几次用洗涤剂丰富的缓冲剂帮助释放细胞内的脂肪碎片, 从而提高产量。根据本协议, 从单个小鼠中消化两个全长坐骨神经, 产生≥30,000 单核事件, 至少3次从根背根中检索到这个数字。CD45 的+白细胞的比例约5% 的总细胞含量的坐骨神经消化和约5-10% 在根背根消化. 坐骨神经中的大多数 CD45+细胞表达了巨噬 CD68 和 CD206。

<table><tbody><tr><td>C57BL/6 Mouse</td><td>Charles River</td><td>C57BL/6NCrl</td><td></td></tr><tr><td>DMEM (+1g/L glucose, Glutamine, Pyruvate)</td><td>Thermo</td><td>31885023</td><td>The source of this material is not important</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Collagenase Type 4</td><td>Worthington Biochemical Corp., US</td><td>LS004188</td><td></td></tr><tr><td>Deoxyribonuclease (DNase) I from bovine pancreas I</td><td>Sigma-Aldrich</td><td>DN25-1g</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>Foetal Calf Serum (FCS), heat inactivated</td><td>Sigma-Aldrich</td><td>F4135</td><td></td></tr><tr><td>Antibody against CD11b-PerCP/Cy5.5</td><td>Biolegend</td><td>101228</td><td>Clone M1/70</td></tr><tr><td>Antibody against MHC class II, biotinylated</td><td>Biolegend</td><td>107603</td><td>Clone M5/114.15.2</td></tr><tr><td>Antibody against CD45-A647</td><td>Biolegend</td><td>107603</td><td>Clone 30-F11</td></tr><tr><td>Antibody against CD68-APC</td><td>Biolegend</td><td>137007</td><td>Clone FA/11</td></tr><tr><td>Antibody against CD206-PE</td><td>Biolegend</td><td>141706</td><td>Clone C068C2</td></tr><tr><td>Antibody against F4/80-PE/Cy7</td><td>Biolegend</td><td>123113</td><td>Clone BM8</td></tr><tr><td>Streptavidin-PE/Cy7</td><td>Biolegend</td><td>405206</td><td>Used to detect MHCII-biotin with CD45-A647 and CDllb-PerCP/Cy5.5</td></tr><tr><td>Streptavidin-PerCP</td><td>Biolegend</td><td>405213</td><td>Used to detect MHCII-biotin with CD68-APC and F4/80-PE/Cy7</td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542-1MG</td><td>Dilute in PBS to a 50x and store at 4 &amp;deg;C in the dark</td></tr><tr><td>Cell dissociation sieve - tissue grinder kit</td><td>Sigma-Aldrich</td><td>CD1</td><td></td></tr><tr><td>V-Shaped, 96-well plates</td><td>Greiner/Sigma</td><td>M8185</td><td></td></tr><tr><td>5ml polystryene round-bottom tube, 12x75mm</td><td>BD Biosciences</td><td>352008</td><td></td></tr><tr><td>60x15mm petri dish</td><td>Greiner/Sigma</td><td>Z643084</td><td></td></tr><tr><td>BD LSR II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

analysis of immune cells in single sciatic nerves and dorsal root ganglion from a single mouse using flow cytometry

神经驻留免疫细胞在周围神经系统 (三七) 是必不可少的保持神经元完整的健康神经。三七总皂苷的免疫细胞受损伤和疾病的影响, 影响神经功能和再生能力。神经免疫细胞通常通过免疫荧光 (如果) 进行分析。如果是确定神经的免疫细胞位置的必要条件, 如果只是半定量的, 并且方法仅限于可以同时分析的标记的数量和表面表达的程度。本研究采用流式细胞术对单个小鼠坐骨神经或背根节 (DRGs) 的白细胞浸润进行定量分析。用 DAPI 进行单细胞分析, 同时分析了几种蛋白质的表面或细胞内表达。根据本议定书处理的一只老鼠的坐骨神经都产生了≥3万单核事件。坐骨神经中白细胞的比例由 CD45 表达, 在坐骨神经中的总细胞含量约为 5%, 在根节中约5-10%。虽然本协议主要关注的是在三七总皂苷内的免疫细胞, 但流式细胞仪的灵活性, 同时测量一些标记, 意味着其他细胞存在于神经, 如雪旺细胞, 周、成纤维细胞和内皮细胞膜, 也可以用这种方法进行分析。因此, 该方法为研究三七总皂甙的系统性影响提供了新的途径, 如神经病变的毒和遗传模型, 以及糖尿病等慢性病。

根据该协议, 从六只健康的 C57BL/6 小鼠中制备了全长坐骨神经和所有主要根背根的细胞悬浮液, 并将其分为三等分染色。反染色与 DAPI, 其中着色 DNA, 允许检测一个单一的细胞数量 (图 1A-B, 上部面板)。冲洗的 DAPI, 在分析前流式细胞仪, 减少到一定程度上的非核碎片的荧光强度, 从而允许选择一个离散的人口在 FSC 轴的单核事件?...

Watch this Scientific Journal Video about Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry at JoVE.com

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

由于组织的缺乏, 小鼠坐骨神经内细胞含量的定量分析是困难的。本协议描述了一种组织消化和制备的方法, 它提供了足够的细胞, 用于对单个小鼠神经的免疫细胞进行流式细胞仪分析。

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 ...

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Research

JoVE Journal

Neuroscience

11.4K 次观看.  University Hospital of Heidelberg. 由于组织的缺乏, 小鼠坐骨神经内细胞含量的定量分析是困难的。本协议描述了一种组织消化和制备的方法, 它提供了足够的细胞, 用于对单个小鼠神经的免疫细胞进行流式细胞仪分析。

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

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 ...

科研

免疫与感染

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

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

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 ...

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 ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

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 ...

神经科学

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 ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

生物工程

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.