Name: visualizing macrophage extracellular traps using confocal microscopy
Uploaded: 2017-10-19T14:00:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Visualizing Macrophage Extracellular Traps Using Confocal Microscopy at JoVE.com

这项工作的经费来自蒙纳士大学、国家卫生和医学研究理事会以及蒙纳隆和睡眠研究所的赠款。作者要感谢在蒙纳士健康, 朱迪卡拉汉, 亚历克斯富尔切, Kirstin Elgass 的临床免疫学工作人员和蒙纳登 Lo 的微成像 (mmi) 的帮助与共聚焦显微镜成像, 和作者承认的 MMI 设施蒙纳士大学。作者承认的设施, 和科学和技术援助的蒙纳的组织学平台。

本协议遵循以下的实验: (1) 人类由蒙纳士医学中心伦理委员会和 (2) 动物的伦理委员会的墨尔本大学. 
 1. 支气管肺泡灌洗 (球) 巨噬细胞 <ol> <li> 使用球获得肺巨噬细胞: (1) 经支气管镜检查的人体受试者 6 , (2) 小鼠使用 intra-tracheal 吸入 13. 
	注: 巨噬细胞的获得通常会引起一些活化作用。巨噬细胞是从需要支气管镜检查, 以调查潜在的医疗问题的病人, 而不是所有的科目可能是适合的分析 (这将需要为每个单独的项目确定)。
	<ol> <li> 采取的解决方案和降速 (500 x g 为10分钟) 形成一个小球, 用培养基洗涤两次 (罗斯威尔公园纪念研究所培养基 (RPMI), 5% 胎小牛血清和 1% l-谷氨酰胺) 在室温下, 然后在4毫升培养基中稀释细胞。嗯. </li>
		<li> 如果需要, 请求正式的差异计数; 大多数单元格 (一般和 #62; 80%) 将是巨噬细胞 6 . 
		注意: 这通常由临床组织学服务使用不同的细胞的形态学出现类型 6 . </li>
		<li> 使用台盼蓝排除和例在球溶液上执行一个可行的巨噬细胞计数. 
		注: 如步骤1.1 所述, 从人和小鼠获得了球溶液. </li>
		<li> 将 1-4 x 10 5 巨噬细胞添加到 24-片中, 并在500和 #181 中的培养基上, 每晚在37和 #176; C; 细胞会附着在片。对于人体样本, 添加抗生素 ( 例如 , 青霉素和链霉素, 10 4 U/毫升), 以防止细菌污染. </li>
	</ol> </li> </ol> 2。巨噬细胞免疫荧光标记/显微术 注意: 如上所述, 细胞附着在片上. 
<ol> <li> 移除培养基, 并用磷酸盐缓冲盐水 (PBS) 清洗细胞一次。在2% 甲醛/高碘酸盐/赖氨酸 (进进) 定影液中固定细胞10分钟. </li>
	<li> 在 pbs 中简要地清洗细胞, 然后在 pbs 中 permeabilize 0.2% 吐温 20, 20 分钟. </li>
	<li> 在 PBS 稀释的5% 牛血清白蛋白 (BSA) 中, 用10% 鸡血清阻断细胞, 30 分钟. </li>
	<li> 在室温下孵育1小时的主、同种控制抗体, 在1:100 浓度下 (更具体的细节列在 材料表中, ) 在37和 #176; C. 
	注: 对于球样, 通常不需要一个特定的巨噬细胞标记. </li>
	<li> 在添加初级抗体后, 在 PBS 中清洗细胞, 并在室温下再用相应的二级抗体孵育40分钟. </li>
	<li> 在405、488、561和 647 nm 和一个40X、1.0 NA 油目标中, 使用共聚焦显微镜 (如上所述) 在 PBS 和芒 DAPI-based 安装介质中清洗剖面以实现可视化. </li>
</ol> 3。肺组织标本 注意: 对于肺组织的 体内 研究, 在相关暴露后 (如 O 和 #39 所描述的那样, 对样本进行研究 ( 如 ); 沙利文 et al. 7 ). 与这个实验最相关的接触是传染性微生物, 特别是细菌。可用于此方法的小鼠菌株为 C57BL/6 或 balb/c/c 型, 10-12 周, 重量 25-30 克, 雄性. 
<ol> <li> 安乐小鼠, 通过腹腔注射氯胺酮 (400 毫克/千克) 和嗪 (40 毫克/千克)。打开胸腔, 用手术钳和剪刀把肺部和心脏暴露出来。
	<ol> <li> 用21口径的针将温热的 PBS 注入右心室, 从血管中冲洗出三十年代的血液, 然后在三十年代注入10毫升的2% 进进的固定液 (进入右心室). </li>
		<li> 使用热 PBS (42 和 #176; C) 冲洗胸腔。气管插管时用21口径的针头和0.8 毫米口径的管子切开, 并注入800和 #181; 5ml (到42和 #176; C), 熔点点3% 琼脂糖溶液. </li>
		<li> 用编织丝 (4.0 USP) 将气管系住。把线绕在气管的插入点下面, 通过一个简单的上手结来保护它。为了固化琼脂糖, 在肺部周围 ice-cold PBS。用剪刀和钝夹层将肺部和心脏从胸腔取出。将每个肺分别取出, 然后在4和 #176 的2% 进工党或福尔马林中固定16小时; C. </li>
		<li> 在4和 #176 将样品储存在 PBS 中, 直到组织切片。这些获得肺组织的方法以前被描述为 13 . </li>
	</ol> </li> <li> 准备肺部组织样本, 请使用以下步骤。
	<ol> <li> 在室温下, 在10% 自然缓冲的福尔马林中修复肺组织 (在步骤3.1 中切除) 组织 (24 h)。转移到75% 乙醇, 并把一个组织处理器在一个12小时的周期, (2.5 小时在75% 乙醇, 3 h 在绝对乙醇, 3.5 h 在溶剂 3B, 并为 3 h 在石蜡). </li>
		<li> 嵌入标准石蜡以创建福尔马林固定的石蜡镶嵌 (FFPE) 块, 它们在室温下存储. 
		注: 在这一阶段, 通常是方便削减肺部部分标准幻灯片. </li>
		<li> 在 4-5 和 #181 上切割 FFPE 肺部分; m 厚度使用切片和安装在带电的、涂覆的幻灯片上, 如前面所描述的 O 和 #39; 沙利文 et al. 7 </li> <li> 装入幻灯片, 在 60 mm x 24 mm 片 (大小 1.5) 上放置3滴安装介质, 反转片上的幻灯片, 并推出多余的介质。密封与指甲油和储存在室温下. </li>
	</ol> </li> </ol> 4。肺组织标本的制备/显微术 <ol> <li> 以脱、水化和预处理 FFPE 肺组织标本和抗原检索溶液。
	<ol> <li> 烘箱在60和 #176 处烘干 FFPE 幻灯片60分钟;然后将二甲苯溶液中的玻片放在30分钟内, 在室温下转移到70% 乙醇溶液中5分钟。用自来水冲洗. </li>
		<li> 在耐高温塑料包装和受 HIER 抗原检索在压力锅为10分钟10米/摩尔/升三, 1 摩尔/EDTA pH 9.0。冷却20分钟, 在自来水中洗两次, 5 分钟在一个摇滚歌手, 然后一次与 PBS 的摇滚. </li>
		<li> 在室温下以5% 的 BSA/PBS 为30分钟的10% 鸡血清块. </li>
	</ol> </li> <li> 染色组织。
	<ol> <li> 要定义巨噬细胞中的 MMP-9, 在 1% BSA/PBS 中添加主要抗体 (H3Cit 和 F4/80), 在4和 #176 时为16小时; C 在1/100 稀释 (有关抗体量的具体细节列在 材料表 中)。用 PBS 在摇杆上洗两次5分钟. </li>
		<li> 在室温下以 1% BSA/PBS 为40分钟, 通过孵育与相应的二次抗体实现荧光检测。抗体是: (1) 鸡 anti-mouse 488 (绿色), (2) 鸡肉山羊 594 (红色), 和 (3) 驴 anti-mouse 647 (远红色). </li>
		<li> 用含有 DAPI 的安装介质在 PBS 和安装中清洗切片, 以进行染色质染色. </li>
	</ol> </li> <li> 使用与倒置显微镜相连的共焦激光扫描头获取荧光图像。
	<ol> <li> 激发405、488、561和 647 nm 激光器的准备工作。使用20X 和 #160, 通过单击线路顺序、调配按钮 (2 线平均) 捕获单平面 512 x 512 像素图像; 0.1 na 空气和40X 和 #160; 1.0 na 油目标. </li>
		<li> 每节至少获得十个视图字段, 用于每个结果的分析和数据 (, 即控件的 、10高功率视图 (HFOV), 10 用于受激样本, 等 , 用于每个鼠标). 
		注: 为了获得一个具有代表性的整个领域的样本, 一个矩阵系统可以用来将场划分为不同的区段和为每个不同的样品相同的矩阵用于选择视图字段 ( 例如 , 该字段可分为9节, 从中心和4角, 两个 HFOV 被采取). </li>
	</ol> </li> </ol> 5。三维 (3-d) 成像的大都会 注意: 由于大都会是3维结构, 因此, 通过采用多个 z 堆栈图像进行重组, 可以提供有价值的信息. 
<ol> <li> 节从石蜡块的肺标本, 在200和 #181; m 使用 vibratome 在振幅设置在1.8 毫米和速度 0.1-0.5 毫米/秒. </li>
	<li> 将肺组织与20% 的血清 (10% 胎小牛血清和10% 鸡血清), 然后 permeabilize 在 PBS 补充和3.75% 卫一-X 100 为 7 h 在室温下温和鼓动. </li>
	<li> 在200和 #181 中, 在4和 #176 上染色16小时的部分; C 与相关的主要抗体 (MMP-9、H3Cit 和 F4/80); 离心管中的 L 体积 (抗体浓度列在 "材料表" ) 中. </li>
	<li> 在室温下用相关的二级抗体在1小时内进行孵育, 在 PBS 中清洗3次, 10 分钟. </li>
	<li> 在溶液中将 DAPI (1:5、000) 的肺部部分染色20分钟, 然后在 PBS 中加入显微镜幻灯片. </li>
	<li> 使用倒置共聚焦显微镜 (40x 1.3 NA) 获得荧光图像, 配备 405 nm、440 nm、473 nm、543 nm 和 635 nm 激光器. </li>
	<li> 记录3维 z 堆叠的0.54 和 #181; m 厚度以最佳 z 切片为 40x 1.3 NA 油目标. 
	注: 所使用的软件将根据不同的显微镜而有所不同。 补充文件 1 中提供了一个示例, 说明如何将该软件用于一个显微镜. </li>
</ol> 6。图像分析 注意: 对样品的分析需要使用特定的成像分析软件和示例列在 材料表 中。虽然对结果的分析将取决于所使用的具体程序, 下面列出的要点是重要的. 
<ol> <li> 分析球样本 <ol> <li> 通过选择 DAPI 的蓝色通道并为单元格指定最小大小 (, 如 , #62; 18 和 #181; m 直径) 来定义巨噬细胞的数量。使用相关软件, 测量每个字段的巨噬细胞总数. </li>
		<li> 通过 DAPI 检测到的胞外染色质的存在 (如 、MMP12、PAD2 和 H3Cit) 的表达, 计算具有所满足特征的单元格数. 
		注: 可以分析的标记数取决于可用激光器的数量, 但在理想情况下除了 DAPI 外, 还应包括至少两个其他标记。可用于满足表达式的其他较不具体的参数 ( 例如 、胞外染色质和扩大核的细胞数). </li>
		<li> 要比较 (在控制、烟雾和烟雾/dnasei 处理的组之间) 中的满足表达式, 请分析每个样本中至少有100个巨噬细胞. </li>
		<li> 测量二级抗体和同种的荧光控制水平; 测量每个样本中至少100细胞的荧光。使用标准软件测量每个标记 ( 例如 , H3Cit) 的背景荧光的平均水平. </li>
		<li> 要定义满足表达式, 请将具有典型表型的单元格计数 ( 如 , 胞外染色质与表达 H3Cit 和 MMP9), 并为每个满足, 测量标记的荧光水平 (, 如 , H3Cit 和 MMP9)。如果细胞的染色不高于背景和同种的控制, 则不排除产生纽约大都会队的电池. </li>
		<li> 对于人类的样本, 分析每一个样本中的100细胞的最小值, 使之成为巨噬细胞的百分比。对于小鼠样本, 由于细胞较小, 而大都会更难定义, 所以分析了每个样本的大量细胞 ( 例如 , 200 巨噬). </li>
	</ol> </li> <li> 分析肺组织样本 <ol> 以确定肺组织中是否存在, 使用特定的巨噬细胞标记, 如 F4/80. <li>
		<li> 通过在表达胞外染色质的细胞中使用 F4/80 的共存, 在6.1 节中列出的其他参数来定义肺组织的存在. </li>
		<li> 确定具有二级抗体和同种控制的样品的背景荧光水平。从不高于背景的分析中排除 "大都会". </li>
	</ol>
	</li>
</ol>

<ol>
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T., 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=Statins+enhance+formation+of+phagocyte+extracellular+traps.">Statins enhance formation of phagocyte extracellular traps.</a> Cell host &amp; microbe. 8 (5), 445-454 (2010).</li><li>Wong, K. W., Jacobs, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+exploits+human+interferon+gamma+to+stimulate+macrophage+extracellular+trap+formation+and+necrosis.">Mycobacterium tuberculosis exploits human interferon gamma to stimulate macrophage extracellular trap formation and necrosis.</a> J Infect Dis. 208 (1), 109-119 (2013).</li></ol>

作者没有对这项工作作出披露。

本评价中描述的方法基于用于定义网的存在14。巨噬细胞是到目前为止的主要单元类型在球样品和这种收集方法特别适合研究大都会。如果在球中存在红细胞, 则应使用氯化铵裂解这些细胞。该球的程序将一般激活巨噬细胞, 因此预计将有 un-stimulated 样本存在。巨噬细胞通常是非常粘附的。由于这些细胞是主要的细胞类型, 而且它们的形态也可以区分, 因此, 它们通常不需要特定的标记。通常在一个球, 大于80% 的细胞是巨细胞 (和嗜中性白细胞少于 5%), 并在多次洗涤后, 通常只有黏附的白血球 (即, 巨噬细胞) 保持6。如果人们对区分巨噬细胞和嗜中性粒细胞的能力有疑问, 可能会使用中性粒细胞弹性蛋白酶等嗜中性细胞标记。在肺组织中, 一个特定的巨噬细胞标记 (例如, F4/80) 是需要的, 因为巨噬细胞很难区别于其他类型。巨噬细胞有荧光, 所以使用背景和同种控制是重要的;这是特殊情况与被刺激的样品。
最好的方式来确定存在的大都会仍有待确定。特定的通路/过程, 这是共同的中性粒细胞和巨噬细胞是: (1) 粒状蛋白酶, (2) citrullination 的组蛋白, 和 (3) 垫。PAD4 更具体的中性粒细胞, 而 PAD2 是更突出的单核/巨噬细胞。F4/80 的使用是小鼠巨噬细胞的接受标记物, 但人类巨噬细胞没有如此明确的标记, 这可能会在评估人类肺组织以满足表达时造成问题。在肺组织中, 由于有许多其他的细胞类型, 而大都会被推到肺泡壁上, 因此分析在肺部的表达可能更加困难。在分析组织切片时, 在横断面平坦时, 最佳的可视化。如果大都会是在一个不同的平面, 可能很难确定是否有一个满足的存在。这个问题可以在一定程度上通过使 z 栈, 使更深层次的组织 (3 维) 的飞机, 以检查大都会和潜在的可视化大都会的不同方面。
没有一种接受的方法来定义肺样的存在。所描述的方法是基于那些用于可视化网。周et al.描述了丝裂原 PMA 诱导的巨噬细胞系, 由存在胞外染色质15评估。随后的一项研究表明,结核分枝杆菌诱导的体外的,由细胞外染色质和 H3Cit16的存在定义。我们在肾小球肾炎的模型中显示了在体内, 这是由 co-localized 染色质、组蛋白和过氧化物7所定义的肾脏中存在的。最后, 我们已经证明了在人肺巨噬细胞的存在, 由细胞外染色质和 MMP12 的结合 6。我们的方法可以被其他研究者用来研究炎症反应的这一重要方面。
很可能, 大都会在慢性炎症性疾病中具有重要作用。导致 NETosis 的进程仍未定义良好。对大都会的研究可为陷阱形成机制提供重要的见解。与更多的标记和功能研究的协议的发展将进一步确定在疾病中的作用。
我们已经在本协议中确定了一些关键步骤。人类的样本可能有次生细菌污染, 在培养基中使用抗生素对限制污染很重要。主要和次要抗体在他们的荧光可能变化, 甚而与同一个制造商。最后, 肺组织标本的分析比球样更复杂, 需要时间来制定一个标准化的方法。

中性粒细胞胞外陷阱 (网), 首先由 Brinkmann et al.描述1它们主要是针对感染 (特别是细菌) 而产生的, 在主机防御中具有重要作用12。他们也被描述为对非传染性疾病, 包括血管炎和系统性红斑狼疮 (SLE) 的反应发生;并对丝裂原佛波 12-myrisate 13 乙酸 (PMA)2,3。最近已经认识到, 其他细胞类型也可能产生胞外陷阱, 包括巨噬细胞。巨噬细胞胞外陷阱 (纽约大都会) 在文献4,5中还不是一个定义良好的实体。我们最近建立的方法, 以检测的存在, 两个在体外和在体内6,7。本文将介绍用共焦显微镜测量大都会的方法。
与其他细胞通路 (如细胞凋亡8) 相区别的 NETosis 的主要特征是染色质的挤压与: (1) 组蛋白的 citrullination (H3Cit)9, (2) 颗粒蛋白酶的表达,10和 (3) 参与肽精氨酸 deiminase (PAD) 411,12。巨噬细胞也表达 H3Cit, 颗粒蛋白酶, 和垫, 这些功能可以用来定义的存在, 大都会。
大都会在肺中具有特别重要的作用, 因为巨噬细胞是肺肺泡和气管中的主要干细胞, 在引导细胞免疫应答感染/炎症方面起着最初的作用。此外, 虽然肺的大部分是空的空间 (例如, 在肺泡内), 大都会有可能扩展到可用的空间, 与固体器官形成对比。
最广泛使用的方法来定义存在的网是通过共焦显微镜。目前还没有一个明确的方法来衡量纽约大都会队。测量网的技术被适应了测量在这个协议的存在的大都会。该方法的主要要求是获得共焦显微镜和适当的成像软件进行分析。

<table><tbody><tr><td>&lt;strong&gt;Human samples: Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rabbit anti-human H3Cit (Citrulline R26)</td><td>Abcam</td><td>AB5103</td><td>10 ug/ml</td></tr><tr><td>Rabbit anti-human MMP12</td><td>Novus Biological</td><td>NB110-57214</td><td>1:100 concentration not quantifiable</td></tr><tr><td>Mouse anti-human MMP9</td><td>Novus Biological</td><td>AB119906</td><td>1:200 ascites, no concentration given</td></tr><tr><td>Rabbit anti-human PADI2/PAD2</td><td>Abcam</td><td>AB16478</td><td>7 ug/ml</td></tr><tr><td>Mouse anti-human PADI4/PAD4</td><td>Abcam</td><td>AB128086</td><td>10.1 ug/ml</td></tr><tr><td>Sheep anti-human NE</td><td>LifeSpan Bioscience</td><td>LS-B4244</td><td>25 ug/ml</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mouse samples: Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Goat anti-mouse MMP9</td><td>Abcam</td><td>AF909</td><td>10 ug/ml</td></tr><tr><td>Rabbit anti-mouse H3Cit (Citrulline R26)</td><td>Abcam</td><td>AB5103</td><td>10 ug/ml</td></tr><tr><td>Rat anti-mouse F4/80</td><td>In-house (hybridoma)</td><td>In house</td><td>20 ug/ml</td></tr><tr><td>Sheep anti-human Anti-HNE /NE</td><td>Sapphire Bioscience</td><td>LS-B4244</td><td>25 ug/ml</td></tr><tr><td>Mouse anti-human &amp;shy;&amp;shy;&amp;shy;&amp;shy;PADI4/PAD4</td><td>Abcam</td><td>AB128086</td><td>10.1 ug/ml</td></tr><tr><td>Super-frost plus slides</td><td>Menzel</td><td>S41104A</td><td></td></tr><tr><td>Dapi-prolong gold</td><td>Molecular probes</td><td>P36931</td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>85111</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>E9434</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chicken anti-rabbit AF 488/</td><td>Life technologies</td><td>A-21441</td><td></td></tr><tr><td>Chicken anti-rabbit AF 594</td><td>Life technologies</td><td>A-21442</td><td></td></tr><tr><td>Chicken anti-goat AF 594</td><td>Life technologies</td><td>a-21468</td><td></td></tr><tr><td>Chicken anti-mouse AF488</td><td>Life technologies</td><td>A-21200</td><td></td></tr><tr><td>Donkey anti-sheep AF 594</td><td>Life technologies</td><td>A-11018</td><td></td></tr><tr><td>Chicken anti-mouse AF 647</td><td>Life technologies</td><td>A-21463</td><td></td></tr><tr><td>Donkey anti-sheep AF 594</td><td>Life technologies</td><td>A-11016</td><td></td></tr><tr><td>Isotype control</td><td></td><td></td><td></td></tr><tr><td>Rabbit IgG</td><td>In house</td><td></td><td></td></tr><tr><td>Rabbit IgG</td><td>In house</td><td></td><td></td></tr><tr><td>Mouse IgG1</td><td>BD Bioscience</td><td>550878</td><td></td></tr><tr><td>Rabbit IgG</td><td>In house</td><td></td><td></td></tr><tr><td>Mouse IgG2a</td><td>BioLegend</td><td>400201</td><td></td></tr><tr><td>Sheep IgG</td><td>In house</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software Programs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Image J</td><td>NIH</td><td></td><td></td></tr><tr><td>To average intensity of fluorphores a standard office application like Microsoft Excel can be used</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C1 Confocal scanning microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>FV1200 Confocal scanning microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tissue sources&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human BAL samples from the bronchoscopy suite at Monash Medical Centre</td><td></td><td></td><td></td></tr><tr><td>Mouse BAL samples and lung tissue from the Department of Pharmacology, University of Melbourne.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI</td><td>Sigma-Aldrich</td><td>r8758</td><td></td></tr><tr><td>Fetal calf serum</td><td>Sigma-Aldrich</td><td>F0926</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sudan black</td><td>Sigma-Aldrich</td><td>199664</td><td></td></tr><tr><td>paraformaldehyde/periodate/lysine (PLP) fixative</td><td>Sigma-Aldrich</td><td>27387</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>214736</td><td></td></tr><tr><td>Ketamine</td><td>Sigma-Aldrich</td><td>K2753</td><td></td></tr><tr><td>Natural formalin</td><td>Sigma-Aldrich</td><td>42904</td><td></td></tr><tr><td>Paraffin</td><td>Sigma-Aldrich</td><td>327204</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A2576</td><td></td></tr><tr><td>Solvent 3B</td><td>Hi-Chem</td><td>2026</td><td></td></tr><tr><td>Coverslips 12 ml</td><td>Sigma-Aldrich</td><td>S1815</td><td></td></tr><tr><td>Coverslips 60 x 24 ml</td><td>Sigma-Aldrich</td><td>C6875</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>c57 black 6</td><td>Monash animal research platform (MARP)</td><td></td><td></td></tr><tr><td>BALB/c</td><td>MARP</td><td></td><td></td></tr></tbody></table>

visualizing macrophage extracellular traps using confocal microscopy

一种用于确定中性粒细胞胞外陷阱 (网) 存在的主要方法是共焦显微镜。我们已经修改了建立的共焦显微镜方法来可视化巨噬细胞胞外陷阱 (纽约大都会)。这些胞外陷阱的定义是存在的胞外染色质与表达的其他成分, 如颗粒蛋白酶, 氨组蛋白, 和肽酸 deiminase (PAD)。一般在接触刺激后测量, 与 un-stimulated 样本相比, 大都会的表达。样品也包括背景和同种控制。使用定义良好的图像分析软件分析单元。共聚焦显微镜可用于定义的存在的两个体外和在体内肺组织。

可从球样, 肺组织和较厚的肺切片中, 3 维图像可视化。在图 1中显示了一个在一个球样中可视化的大都会示例。大都会的形态学根据其成熟阶段而变化。显微镜上第一个可探测的特征是细胞核在细胞边缘的运动。其次是胞外染色质与其他 co-expressed 介质, 如 H3Cit 和颗粒蛋白酶。在早期阶段, 细胞仍然有一个大致球形的形状与胞外陷阱表达。在以后的阶段, 遇见的形成以细胞的身体的伸长为特征。
肺组织的显示在图 2。当肺部被液体膨胀以定义肺部结构时, 大都会被推向肺泡壁。大都会的形态也会因组织被切割的平面而异。用于肺组织标本的标准切片厚度为4-5 µm。较厚的组织切片可使多个图像被拍摄, 然后可以在3维中查看。抗体只会渗入到肺组织中, 而25-50 µm 的切片厚度是最佳的。3维图像的一个示例显示在图 3和视频 1中。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56459/56459fig1.jpg"> 
图 1:球会。人类的大都会在不同发育阶段的刺激后形成。表达 H3Cit 和 MMP9 的胞外染色质特征。染色 (A) 染色质, (B) H3Cit, (C) MMP9, 和 (D) 合并图像。面板插入是同种控件。缩放条形图 = 10 µm (15 µm 同种)。在右上角显示了一个较早的阶段, 大致呈球形。一个更成熟的相遇显示在田野的中间。使用激光扫描共聚焦显微镜和 40x 1.0 NA 油目标拍摄图像。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56459/56459fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56459/56459fig2.jpg"> 
图 2: 肺组织.刺激后的肺组织中的小鼠。H3Cit、MMP9 和 F4/80 (作为巨噬细胞标记) 的胞外染色质具有表达的特征。面板 (A) 显示了一个典型的高功率视场, 用于测量纽约大都会的数量;面板 (B-F) 显示更高放大倍数下的虚线正方形区域。染色 (B) 染色质, (C) H3Cit, (D) MMP9, (E) F4/80, 和 (F) 合并图像。"插入" 面板是同种控件。缩放条形图 = 20 µm (20 µm 同种)。大都会被推到肺泡壁上使用激光扫描共聚焦显微镜和 40x 1.3 NA 油目标拍摄图像。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56459/56459fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56459/56459fig3.jpg"> 
图 3: 肺部部分的三维视图.小鼠肺组织固定, 切成25-35 µm 厚切片。在1µm 厚度的切片上, 组织被标记为纽约大都会和影像。图像与 z 堆栈组合, 以创建一个3维的图片的 MET。使用激光扫描共聚焦显微镜和 40x 1.3 NA 油目标拍摄图像。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56459/56459fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Video 1" src="/files/ftp_upload/56459/56459video1.jpg"> 
视频 1: 肺部部分的三维视图.视频对应于图 3中提供的示例。轴刻度 = 5 µm.<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56459/Mets_1.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

Watch this Scientific Journal Video about Visualizing Macrophage Extracellular Traps Using Confocal Microscopy at JoVE.com

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

巨噬细胞胞外陷阱是一个新描述的实体。这篇文章将集中于共焦显微镜方法和如何他们是可视化的体外和体内从肺部样本。

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

visualizing-macrophage-extracellular-traps-using-confocal-microscopy

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

10.1K 次观看.  Monash Medical Centre. 巨噬细胞胞外陷阱是一个新描述的实体。这篇文章将集中于共焦显微镜方法和如何他们是可视化的体外和体内从肺部样本。

视频: Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

微生物和化学刺激诱导的中性粒细胞胞外陷阱的形态学和组成分析

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

医学

染色和成像以可视化中性粒细胞胞外陷阱

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

作者聚焦：人和小鼠组织中的中性粒细胞胞外陷阱成像 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...

In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

Source: Zhang, Y., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60541">In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages.</a> J. Vis. Exp. (2019).
This video demonstrates in vitro stimulation of macrophage extracellular traps (METs) in human monocyte-derive macrophages using different inflammatory stimuli such as TNF-alpha, PMA, and HOCL. The video also demonstrates the method of SYTOX dye staining of METs for visualization.

巨噬细胞细胞外陷阱的体外刺激和可视化

1. Polarization of HMDM

	Under sterile conditions, prepare the M1 priming media by adding interferon &#947; (IFN&#947;; 20 ng/mL) and lipopolysaccharide ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids

Source: Sharma, R. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56459">Visualizing Macrophage Extracellular Traps Using Confocal Microscopy.</a> J. Vis. Exp. (2017)
This video demonstrates the immunolabeling of macrophage extracellular traps, METs, in bronchoalveolar macrophages from bronchoalveolar lavage fluid, and their visualization using a confocal microscope.

人支气管肺泡灌洗液中巨噬细胞细胞外陷阱的可视化

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Bronchoalveolar Lavage (BAL) ...

利用共焦显微镜可视化巨噬细胞胞外陷阱

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此视频中的章节

嗜中性粒细胞胞外的陷阱：如何生成和可视化

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

微生物和化学刺激诱导的中性粒细胞胞外陷阱的形态学和组成分析

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

巨噬细胞细胞外陷阱的体外刺激和可视化

人支气管肺泡灌洗液中巨噬细胞细胞外陷阱的可视化