作者想感谢帮助优化仔细阅读了手稿的活体电影，乔纳森博士林德基斯特拉尔斯Philipsen博士，并在方法的发展进行有益的探讨和意见Gunzer实验室的所有成员。这项工作是由补助金爵从德意志研究联合会（DFG SFB 854）

1。感染<ol><li>肿胀分生孢子模具曲霉 （菌株ATCC 46645）的RPMI 1640（Biochrom AG，德国柏林），辅以预培养产生的5％FCS（V / V ）。 2X10 7休息孢子悬浮于5毫升培养基和培养在6孔组织培养板（TPP的公司; Trasadingen，瑞士，产品编号92006）在37 ° C为7H </li><li>肿胀期之后，荧光标记的含6孔加入10μLcalcofluor白原液（西格玛Aldrich公司，Deisenhofen，德国，产品编号F3543; [25]在DMSO毫克/毫升）每个曲霉孢子板，达成最终的calcofluor浓度为50微克/毫升的RPMI。温柔的搅拌后，孢子在37℃孵育15分钟的额外</li><li>在接下来的步骤，孢子是通过过滤收获，通过一个70微米的细胞过滤器（BD公司，德国海德堡）。一次用10 ml PBS在室温下5分钟）（900xg离心洗涤后的沉淀悬浮于2毫升PBS总数计数与纽鲍尔室确定肿分生孢子。最后孢子悬液纺900xg在室温下5分钟，小心取出上清液，沉淀悬浮终浓度为每100μLPBS 1x107孢子。 </li><li>感染，100μL孢子的解决方案是适用于气管内到雌性小鼠（C57/BL6 8-10周龄，哈伦，德国）。如果中性粒细胞以及待观察，赖氨酸EGFP小鼠的使用，携带下溶菌酶 1启动子的EGFP的建议。首先麻醉与氯胺酮150μL/ Rompun解决方案（Inresa医药有限公司，弗赖堡，德国（氯胺酮）和拜耳生命有限公司，勒沃库森，德国（Rompun），1毫升氯胺酮50 mg / ml的单一IP注射动物+ 0.5毫升Rompun [2％] + 3.5毫升无菌氯化钠[0.9％]）。 5分钟后麻醉鼠标可以是固定的，与它的牙齿松紧带的斜坡（即一个倾斜的表面），以方便插管（补充图1）。使用镊子，舌头被拉到一边和一个22G留置静脉留置针（德国贝朗公司，Melsungen，Vasofix Braunüle），可轻轻插入进气管与鹅颈灯永久照明。与机械通气的频率，通过观察动物的胸部经常运动的导管插入成功验证。当已被证实的成功插管，孢子悬液100μL适用于使用100μL微量。此卷应在1-2秒没有任何额外的帮助动物吸入加强内肺部真菌粒子分布是通过机械通风2分钟，用小动物呼吸机的速度每分钟250呼吸的吸入量和300（MiniVent，雨果公司，德国）3月Hugstetten，受感染的动物每呼吸μL（补充图2）。感染后动物都回到自己的笼子，每5分钟再观察，直到日间。老鼠不显示在以下潜伏期疼痛或痛苦的任何证据。 </li></ol> 2。龙准备<ol><li>孢子感染后7小时，动物安乐死过量的异氟醚（Unterschleiβheim，百特公司，德国），直至运动和呼吸停止超过30秒。随后是作为一种辅助方法，以保证临床死亡，使气管和肺部的可视化和访问的开胸手术。然后小心打开，露出胸部的肺和上呼吸道。然后另一个22G留置静脉导管插入气管暴露会厌开始。通过这个小管的肺部充满了1毫升预热低熔点琼脂糖使用1毫升Omnifix的注射器（贝朗）（W / V，2％Promega公司，曼海姆，德国）。只要肺部充满气管打结与缝纫线的一小段，整个动物被放入冰箱，在4 ° C </li><li> 15分钟后，琼脂糖已经凝固时，全肺切除气管开始。用一双锐利的尖剪刀，右肺叶随后被删除，底面是简要干上一张纸巾，然后整个叶粘编制一个vibratome（坎普登仪器，英国，752M Vibroslice块）使用组织胶固定（卡尔罗斯公司，卡尔斯鲁厄，德国，ROTI中学1）下降。 1分钟后，该块安装在切割室，这是充满预冷（4℃），PBS的vibratome。 vibratome是一个中档的振动（5≤10）和速度适中（5≤10。）这样的横截面肺是产生。上半部分的器官，然后转移到一个小的塑料培养皿（直径5厘米的），它是倒一个自制平垫圈（1厘米内径），是一套并行的尼龙线，覆盖固定，除了每1毫米（补充图3）。最后培养皿中充满了10毫升PBS 10μL的DNA染料Sytox橙[5毫米（Invitrogen公司，德国），在最后5微米的染料浓度补充。 </li></ol> 3。双光子激光显微镜<ol><li>培养皿中含有肺样本，然后安装在显微镜下的目标，使缓冲区可升温至37 ° C的温度传感器控制的加热器（补充图4）。双光子显微镜，然后进行了全切一个堂堂正正的Axio上考官配备一个20xNA1.0水浸渍镜头（蔡司耶拿，德国）的阶段使用的Zeiss LSM 710非线性光学显微镜的表面。不同地区，沿节成像扫描检测绿（530纳米）和红色（580 nm）的荧光，以及第二次谐波产生（SHG）信号和照明波长800纳米到400微米的深度使用calcofluor蓝色荧光（在400-470 nm发射）与外部非退扫描探测器（NDD）。除了2 - D图像和时间的推移电影（1图像每隔5-10秒），可单一或重复的3维图片栈随后呈现为体素的效果图或单个扩展的重点图像或使用2,4 - D数据（随着时间的推移3D）不同的软件包。这种微观设置允许在一个几乎完整的环境，因为它是各种空气中的病原体入境口岸，巨大的免疫相关性的细胞行为的观察。原位条件下，细胞运动，细胞的吞噬功能和内源性感染中性粒细胞与模具曲霉的净产量可以很容易地根据这些调查。 </li></ol> 4。代表性的成果如果做得正确的成像会产生2 - 或3色的幻灯片或电影。虽然颜色是在后处理过程，因此是自由的适应性，我们一般选择配色方案，反映了自然色的染料/信号，这是相对的通道检测。因此，Sytox染料描绘红，真菌以及倍频信号以蓝色显示，目前，EGFP标记细胞在绿色染。在第一个例子（图1）蓝色的SHG信号描绘非肺部感染的组织纤维和红色Sytox信号是由居民肺细胞的细胞核产生的vibratome削减开放。在第二个例子（图2）所示，肺部感染，肺泡结构以及真菌群众，以蓝色显示，而原子核和以英语为母语的英语教师都染成红色。第三个例子（图3）是从赖氨酸- EGFP的转基因动物，除了蓝色和红色的结构绿色的中性粒细胞也可见到。补充电影的中性粒细胞和吞噬时间的推移序列中的个别真菌元素的迁移。 <img src="/files/ftp_upload/2659/2659fig1.jpg" alt="图1" /> 图1。在2色的非感染性肺切片的外观，双光子显微镜。一个肺片准备从非感染C57/BL6鼠标成像在协议中所述。这里呈现的是肺泡组织结构（a）的倍频信号，在肺片（B）和覆盖的两个通道（三）编制的细胞核Sytox信号剖开。盒装（三）（四）扩大面积。请注意，在肺底部的纤维组织切片相比，上述肺呼吸活跃的领域内明显的肺泡组织。 <img src="/files/ftp_upload/2659/2659fig2.jpg" alt="图2" /> 图2。从感染7 H一个C57/BL6小鼠的肺切片前与 A在2 色曲霉菌感染的野生型动物的肺切片双光子显微镜的外观。 霉菌是准备和成 ​​像在协议中所述。显示的是合并真菌结构和SHG（一）肺泡组织，Sytox信号的DNA以英语为母语的英语教师，以及细胞核削减编制的肺片（二）期间开放，和两个通道的叠加（三） 。盒装（三）（四）扩大面积。请注意，真菌群众，肺泡和网络结构明显不同的地区都以字母F，A和N，分别标记。 <img src="/files/ftp_upload/2659/2659fig3.jpg" alt="图3" /> 图3。的曲霉菌感染赖氨酸- EGFP动物的肺切片的3种颜色的外观，双光子显微镜。一个一个赖氨酸EGFP小鼠的肺切片感染之前7小时曲霉和成像在协议中所述。显示的是真菌的结合结构和SHG肺泡组织（蓝色），细胞核和以英语为母语的英语教师（红色）Sytox信号，以及以及众多的中性粒细胞（绿色）。 <img src="/files/ftp_upload/2659/2659supfig1.jpg" alt="图4" /> 参考图1。鼠标插管固定。氯胺酮/ Rompun麻醉下的动物是固定的松紧带，在它的牙齿，以便与22G留置静脉导管插管。 <img src="/files/ftp_upload/2659/2659supfig2.jpg" alt="图5" /> 参考图2。机械鼠标的通风。插管鼠标感染1 × 10 7孢子在100μLPBS重悬它的应用程序。加强内肺部真菌粒子分布是通过机械通风与小动物呼吸器感染小鼠。 <img src="/files/ftp_upload/2659/2659supfig3.jpg" alt="图6" /> 参考图3。肺叶内固定准备右肺叶后的器官是利用实验室的平垫圈，是一套并行的尼龙线，覆盖固定在培养皿。 <img src="/files/ftp_upload/2659/2659supfig4.jpg" alt="图6" /> 参考图4。双光子成像设置。培养皿中含有右肺叶被安装在加热垫后的双光子显微镜下的DNA染料Sytox橙此外。 参考电影：中性粒细胞的迁移和吞噬曲霉菌感染肺部真菌因素，如时间的推移看到一个活生生的肺切片7 h后真菌感染的双光子显微镜中性粒细胞是绿色的，真菌的元素，倍频是蓝色，细胞核作为以及网络结构被描述为红色。实验的实时显示在右下角。比例尺描绘50微米。 <a href="/files/ftp_upload/2659/5_9_1 - supplemental.mpg">点击这里观看视频</a>

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实时双光子显微镜在体内或在完整的器官已经获得了在处理与免疫细胞的生理机能的研究在过去10年的深刻重要性。正是有了这种技术，首次成为重要事件，如淋巴结内的T细胞激活的动态可见2-4。最近，研究人员也开始分析特定的细胞功能的效应细胞在淋巴组织中使用此方法5代的第一个步骤一样。 然而，尽管一些新的生物概念使用此方法已被揭露出来，也有仍然充满挑战，没有活体可视化研究迄今已发表的重要问题。值得注意的是，这适用于哺乳动物的肺。本机关的有趣的方面，各种空气中的病原体进入端口，使得它在免疫过程在哺乳动物体内发生的最关键的表面之一。每一个在整个寿命的气息，不必要的颗粒被吸入其中一些有可能诱发危及生命的感染 6 。这是不言自明的，在这样一个敏感的和濒危的网站的一个严密的防御机制，网络，需要将目前参展的免疫反应的整个剧目。另一方面，这是非常重要的，严格控制对在这样一个“脏”的地方潜在的病原体诱导免疫斗争。夸张的免疫系统反应的大规模刺激非特异性免疫细胞的动作7,8时受伤的器官组织损害自己的身体承受的高风险。 在这些想法，那将是非常有趣和有用的，有可能在体内条件下，调查真正的细胞的行为，在哺乳动物的肺。然而，事实上，这种制度至今没有得到成功实施清楚地指出已成立了一个工作协议要解决的巨大的困难。最苛刻的挑战可能是集中稳定。肺负责呼吸的器官是在所有三个方向的空间，实现吸入下不断运动。仅这一情况会导致严重的成像问题，也算是一个活体成像仪“噩梦”。维空间中的任意轻微的议案，已经有一个微观的观点，这需要与稳定，以产生有意义的图像 9微米精度的大规模恶化的效果。由于其固有的紧张的地方重点 ，10，双光子显微镜更是敏感的焦点不稳定，只是在Z方向的几微米的范围内，在一定的结构错位是相当于一个重点完全丧失，从而一个失败的实验。 在这项研究中观察免疫细胞在小鼠肺癌的协议仍然没有在体内的应用，而是一个近似在一个功能完整的肺11的情况。成像淋巴细胞的体外方法，例如在explanted淋巴结节点，已被证明产生的结果为true，相当于在体内观察12，因此是高度相关的5。 在肺片，这是我们的做法可能实地观测，在切除后不久，肺部感染的地方。在切割过程中的3D完整的琼脂糖基质，一个必不可少的步骤，以便精确控制切割过程中，肺的保证。虽然是必要的冷却explanted肺短的时间内，让一个凝固的琼脂糖基质，它有可能返回到接近生理条件下，切割和组织复温后的细胞。这清楚地表明我们的数据，这表明，在这些条件下的中性粒细胞非常活跃，并展示其敏捷的吞噬细胞，这是曲霉感染的有效清除所需的全部潜力。他们巡逻的肺组织，通过上皮屏障，以达到肺泡内部分传递，此外，他们积极参与了真菌孢子11。这项工作的一个重要发现是外观类似曲霉菌感染的器官的中性粒细胞（以英语为母语的英语教师）外的陷阱结构。以英语为母语的英语教师是最近发现了一个新的防御机制在中性粒细胞 13 。然而，自2004年最初的描述，已在动物模型或已观察到这一现象或缺乏人类的生理或病理条件下的数量爆炸式增加14-16。有趣的是，这些结构上花了这么多不同的群体，虽​​然这么多的工作已，大多数报告仍然非常描述的水平上，并没有多少了解净释放及其调控机制。随着我们的协议中，我们能够在第一时间出现在肺部感染的净纤维。此外，我们可以证明，刚刚招募的中性粒细胞以及分子真菌结构，其发生或抑制11重要性。这清楚地表明我们的方法的潜力进行调查NET形成更详细的单步。人们可以认为例如使用合适的基因敲除小鼠的中性粒细胞，观察其继转移实验净形成的能力。 因此，虽然从外周血中性粒细胞移民的直接观察是不是由于这个系统后器官explantation的血液供应不足的可能，我们仍然相信，我们的协议是一个有价值的，也比较容易处理的方法，使成像在对肺部感染的免疫防御的早期或晚期的步骤。因此，这是一个对调查范围内的活体动物的肺呼吸这种现象的重要一步。

direct observation of phagocytosis and net-formation by neutrophils in infected lungs using 2-photon microscopy

经过胃肠道，肺，是世界第二大脊椎动物的身体和环境之间的相互作用的表面。在这里，一个有效的气体交换，必须保持下去，而同时避免了在正常呼吸时吸入的多种病原体的感染。为了实现这一目标，存在一个高超的体液免疫和细胞免疫机制相结合的防御战略。急性肺防御的最有效措施之一是招聘的中性粒细胞，，吞噬吸入病原体或杀死他们释放细胞毒性化学物质。最近除了中性粒细胞库是其爆炸释放外，细菌或真菌甚至NET释放细胞死亡后，可以被捕获或灭活的DNA，以英语为母语的英语教师。我们在座的方法，允许一个直接观察到嗜中性粒细胞，在最近肺部感染迁移，吞噬病原真菌以及可视化整个受感染的组织，他们已经产生广泛的以英语为母语的英语教师。该方法描述了7小时后，小鼠气管内感染与模具曲霉和其考试分生孢子多色时间推移双光子显微镜厚可行的肺片的准备。这种方法允许一个直接调查本地肺组织中的抗真菌防御肺免疫力的详细调查，从而打开了新的途径。

Watch this Scientific Journal Video about Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy at JoVE.com

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

我们展示，如何使用双光子显微镜观察中性粒细胞在受感染的肺部的粒细胞动力学，而他们吞噬病原体或产生中性粒细胞（以英语为母语的英语教师）外的陷阱。

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

direct-observation-phagocytosis-net-formation-neutrophils-infected

Research

JoVE Journal

Immunology and Infection

20.6K 次观看.  Otto-von-Guericke University Magdeburg. 我们展示，如何使用双光子显微镜观察中性粒细胞在受感染的肺部的粒细胞动力学，而他们吞噬病原体或产生中性粒细胞（以英语为母语的英语教师）外的陷阱。

视频: Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

科研

免疫与感染

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.