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>Human samples: Primary antibodies</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mouse samples: Primary antibodies</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 &#173;&#173;&#173;&#173;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary antibodies</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software Programs</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tissue sources</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Media</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other reagents</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mice</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/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/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/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="http://ecsource.jove.com/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

Nanyang Technological University

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

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾:寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.