Name: assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy
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这项工作是由国家过敏与感染疾病格兰特R01AI079253（以BHS）的支持，以及由美国国家癌症研究所癌症中心支援津贴CA016056到罗斯威尔公园癌症研究所。

动物在这项研究中执行的所有程序批准了动物护理和使用委员会在罗斯威尔公园癌症研究所和遵守所有州，联邦和卫生法规国家机构。 1， 在体内巨噬细胞标记注意:PHK26是将要采取由吞噬细胞在血液循环和在组织中静脉内给药（IV）时，亲脂性染料。 PKH26用于体内标记，以减少收获后处理的巨噬细胞的数量。 PKH-26具有稳定地积聚在肺泡巨噬细胞的延长期的优势。给药后10天等待允许从循环只留下肺泡巨噬细胞稳定标记PKH26 8,9所有标记细胞的间隙。 PKH26是一个红色的荧光，具有激发（551 nm）和发射（567 nm）的特性兼容罗丹明或藻红蛋白的检测系统。任何标记过程可以介绍的神器，其中包括生存能力的丧失。然而，由于我们使用的巨噬细胞的标记以同一方法，不同的鼠标基因型（ 例如 ，WT与NADPH氧化酶缺陷），这些文物的影响将是统一的。 <ol><li> PKH26稀释的原液（乙醇1×10 -3 m）在由制造商提供的稀释液1:5。 </li><li>负载100μl的稀释PKH26成1/2 20ng/ml胰岛素注射器（29克X 1/2"）对每只小鼠。 </li><li>通过静脉注射（或尾静脉或眶）至少10天，收获前管理到小鼠。 </li></ol> 2，收获肺泡巨噬细胞支气管肺泡灌洗（BAL） 小鼠用于实验的数目可以变化。一个典型的产量从未受刺激的小鼠是在3-5×10 5肺泡巨噬细胞的范围内，但这个数字也相差很大BAS版上的因素，如进行灌洗者的鼠标，经验大小和灌洗液和从肺中排出的体积的滴注的数量。在这个协议中肺灌洗是一个封闭胸腔它伴随着反复1毫升灌洗有助于提高细胞产量进行。 <ol><li>通过使用无菌条件下，保持溶液的无菌管理致死剂量阿佛丁麻醉剂（12.5毫克），腹腔（IP）安乐死鼠标。 </li><li>用70％乙醇彻底喷鼠标。 </li><li>从动物的颅年底去除皮肤暴露胸部（ 图1A）。 <ol><li>做一个小切口在皮肤只是隔膜下方。 </li><li>将手指插入切口，拉皮肤远离动物（ 图1A）的颅结束。 </li><li>向上推略微的头，延长颈部腹侧（ 图1A）。 </li></ol></li><li>通过切左肋一个小孔进入右侧胸廓放气肺和也允许在以下步骤中的肺的可视化。 </li><li>找到气管，并认真剖析了周围的组织。 <ol><li>取出唾液腺，胸骨舌骨肌和喉等用弯钳。 </li><li>小心地抬起了弯钳和切去，用剪刀薄组织覆盖气管（外膜）揭示的基本环状软骨结构。 </li></ol></li><li>将导管插入气管。 <ol><li>用弯钳，放置缝合后面（背侧）的气管，并通过开口拉一个10cm的长度。 </li><li>启动环状（软骨环增厚）上面仔细地引导22政导管下来气管停在那里气管消失胸骨后面。 </li><li>紧紧绑周围缝合气​​管导管到舒适的位置。从导管取出针头。 </li></ol></li><li>装配4路阀。 <ol><li>填1个6毫升注射器用DPBS，1％FBS和附加到4路阀， 如图1B所示。 </li><li>附上一个空6毫升在注射器上的4路阀（ 图1B）所指示的位置。 </li><li>重视开放端口上的活塞，以导管4路（ 图1B）。注意不要从气管导管打跑。 </li></ol></li><li>从收集肺灌洗液。 注:调查人员可以选择使用肺泡灌洗液中下三卷，以尽量减少组织损伤的可能性。那同样的方法在所有小鼠组被贯彻应用是很重要的。 <ol><li>所以转空注射器被关闭的水龙头把手， 慢慢地注入约1mL DPBS，1％FBS膨胀的肺。通过切孔在步骤2.4观察肺胀。 </li><li>要小心，不要过度膨胀的肺。 </li><li>打开水龙头手柄，使之充分注射器被关闭，并仔细撤回流感<html> ID从肺部。 </li><li>观察肺放气通孔切割步骤1.4。导管可能需要移动稍微向后或向前来获得一个很好的借鉴。采取特殊的预防措施，以保持导管气管内。 </li><li>重复步骤2.8.1-2.8.4 5-6倍，直到所有的DPBS，1％FBS的已注入和从肺部排出。注意:肺液的回收将不注射液的100％。 </li></ol></li><li>空注射器灌洗液到50ml锥形管中。保持细胞在室温下。 </li><li>通过在300 XG离心5分钟，在室温下沉淀细胞，并倒出上清液。这样如果需要，可以保存用于细胞因子分析。 </li><li>重悬的细胞在1-5毫升的RPMI 1640含10％FBS和血细胞计数器计数。在未刺激的小鼠，&gt; 95％的细胞由BAL收获的预计是巨噬细胞，这可以通过细​​胞学进行确认。 </li></ol> 3，培养和收获真菌"&gt;在这个协议中使用的烟曲霉表达绿色荧光蛋白在其整个生命周期的各个阶段:孢子，germlings和菌丝这种真菌菌株是由马戈·摩尔博士，西门菲沙大学，伯纳比，不列颠哥伦比亚省，加拿大提供。 <ol><li>在沙氏脑心脏浸液（BHI）琼脂斜面用氯霉素（0.05克/升）和庆大霉素（0.5克/升）表达GFP的烟曲霉菌株的分生孢子盘。 </li><li>在孵育在室温下松瓶盖为7〜10天的黑暗。 </li><li>收获分生孢子通过倾斜洗涤用10毫升0.01％吐温20的DPBS。轻轻刮除木耳与stripette松动。冲洗斜几次，以增加产量。 </li><li>通过100微米的细胞过滤网通过分生孢子悬浮液置于50ml锥形。 </li><li>粒料分生孢子在400×g离心离心10分钟。 </li><li>分生孢子重悬在50毫升的DPBS和计数使用血球。 </li><li>用DPBS洗两次并稀释至所需浓度滤过。 </li></ol> 4，真菌挑战和共聚焦显微镜为了研究巨噬细胞NADPH氧化酶在宿主防御中的作用，从三种基因型控制吞噬曲霉菌孢子的生长小鼠分离的肺泡巨噬细胞的能力进行了评估。在本研究中使用的基因型包括; 1）野生型小鼠，2）NCF1 * / *锰小鼠的天然存在的突变NCF1（NCF1编码为P47 PHOX，吞噬细胞的NADPH氧化酶的必需组分）离开他们的NADPH氧化酶缺陷型，和3）NCF1 * / *锰+转基因小鼠（MN表示该转基因携带野生型NCF1人类CD68启动子的控制下），其中NADPH氧化酶活性已被重新溶解在单核细胞/巨噬细胞群10，11。 共聚焦显微镜最适合于该试验中，由于存在和在幻灯片非吞噬的分生孢子的生长。共焦显微镜将允许在Z轴中的各个平面的可视化，并且使真菌生长围绕，而不是内巨噬细胞之间的差异。所有的共聚焦图像是使用63X油浸镜头获得的。 Z-栈收购与2.5电子的变焦倍数。一个Z-堆栈的厚度是用DAPI和亮场图像的定位字段的顶部和底部的确定。 Z-堆栈介乎14至24微米，个别切片〜1微米厚。巨噬细胞的定量，1X电子变焦的单扫描每基因型的10个字段分别进行。被确定完整的巨噬细胞的基础上分别PKH26和膜和细胞核的DAPI染色。分生孢子的基础上确定的FITC表达和亮场图像。 <ol><li>准备无菌盖玻片和板<ol><li>下一个生物安全柜，浸泡22×22毫米的玻璃盖玻片在70％桥亚乙基升5-10分钟。 </li><li>在无菌PBS冲洗盖玻片。 </li><li>用无菌镊子，轻轻地放在一个单独的盖玻片在无菌单独包装的6孔板的每个孔的底部。 </li></ol></li><li>种子〜1×10 6个收获的PKH26标记的肺泡巨噬细胞悬浮于300微升的RPMI 1640，10％FBS到每个盖玻片。取决于收获产量的确切数量可以改变。 </li><li>让巨噬细胞在37℃温育，用5％的CO 2过夜以粘附。 </li><li>第二天早晨，加〜1×10 7表达GFP的A。曲霉分生孢子悬浮于100μl预热的（37℃）的RPMI 1640，10％FBS。 </li><li>返回板到37℃培养箱中，用5％的CO 2。 </li><li>细胞应该被固定为至少2小时，室温过夜或在4℃下在3,7和14小时修复细胞，加入2％的甲醛等体积到各孔中（最终浓度1％的甲醛）。地方板的在4℃下早期时间点过夜。在最后的时间点（14小时），固定的细胞在室温下搅拌2小时。 </li><li>固定后，使用镊子轻轻取出盖玻片和冲洗的DPBS。 </li><li>放置盖玻片的边缘折叠的Kimwipe去除多余的液体。 </li><li>适用于6微升荧光封固剂用DAPI到玻璃显微镜载片。 </li><li>莱盖玻片的一个边缘上的幻灯片，并与细胞侧轻轻下拉到安装介质。安装介质将蔓延并形成均匀的一层盖玻片下方。有没有必要压在盖玻片。 </li><li>密封盖玻片的边缘与透明指甲油，并在空气中晾干。 </li><li>店准备的幻灯片在幻灯片框（避光），在室温下，直到准备通过共聚焦显微镜来分析。 </li></ol>

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使用这种方法进行体外分析巨噬细胞的抗真菌活性连同体内真菌的挑战，我们先前表明，在巨噬细胞NADPH氧化酶起着抵御A的重要作用烟4。使用隔离的肺泡巨噬细胞使我们能够专注于他们的抗真菌活性在没有其他免疫细胞（ 如中性粒细胞招募）， 在体内防御曲霉 。 各种可视化策略已经被用于评价巨噬细胞的抗真菌活性<e...

肺泡巨噬细胞是遇到吸入微生物一线吞噬细胞。巨噬细胞识别黑图案由病原体识别受体，摄取并限制吸入孢子（分生孢子）的生长，并启动炎症反应。吞噬细胞NADPH氧化酶转化分子氧超氧阴离子和下游活性氧中间体（投资回报）​​。慢性肉芽肿病（CGD）是NADPH氧化酶的特征是严重的细菌和真菌感染和由过度的炎症反应的一种遗传性疾病。 虽然NADPH氧化酶是至关重要的中性粒细胞介导的宿主防御1 - 3，NADPH氧化酶的巨噬细胞中的重要性是不明确的。此前的研究表明，肺泡巨噬细胞摄取并杀死曲霉菌孢子，而中性粒细胞主要针对菌丝阶段5。但是，也出现了相互矛盾的雷苏尔TS作为NADPH氧化酶在巨噬细胞中控制A的生长的作用曲霉孢子6,7。 该方法的目标是界定在介导针对A.宿主防御NADPH氧化酶的巨噬细胞中的具体作用烟曲霉菌孢子。我们发现，在肺泡巨噬细胞NADPH氧化酶控制吞噬A的增长烟曲霉菌孢子4。为了最准确地模拟主机对吸入真菌，我们使用了支气管肺泡灌洗收集肺泡巨噬细胞的未刺激小鼠紧随牺牲。使用隔离的肺泡巨噬细胞使我们能够专注于自己的抗真菌活性在没有其他免疫细胞（ 如中性粒细胞招募）， 在体内防御曲霉 。在该方法中，肺泡巨噬细胞进行染色在事先体内收获，以尽量减少收获后的操作量。&lt;/ P&gt; 另一个优点这个协议是使用GFP表达A的曲霉菌株。这种真菌表达从分生孢子阶段GFP通过菌丝阶段，并没有必要进一步染色。以获得具有更详细的图像，我们选择了共焦显微镜使从所获得的图像的三维结构的重建。三维重建给出菌丝生长周围，而不是在或通过巨噬细胞之间进行区分的能力。分生孢子也可以精确地辨别作为细胞内与细胞外。

<table><tbody><tr><td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td><td>Sigma</td><td>PKH26GL-1KT</td><td></td></tr><tr><td>2,2,2 Tribromoethanol</td><td>Sigma</td><td>T48402</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma</td><td>A-1685</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>Dulbecco&amp;#039;s Phosphate Buffered Saline (without calcium and magnesium)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>A0171</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>P91444</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>22 G x 1.00 inch i.v. catheter</td><td>BD</td><td>381523</td><td>Insyte Autoguard Winged</td></tr><tr><td>Insulin syringes</td><td></td><td></td><td></td></tr><tr><td>6 ml Syringes</td><td></td><td></td><td></td></tr><tr><td>4-way stopcock</td><td>Fisher Scientific</td><td>50-700-077</td><td></td></tr><tr><td>Suture thread</td><td></td><td></td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>gauze sponges (4 inch square)</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td>heat inactivated</td></tr><tr><td>Hema 3 Stain Set</td><td>Fisher Scientific</td><td>22-122-911</td><td></td></tr><tr><td>Cytoseal 60</td><td>Thermo Scientific</td><td>8310-4</td><td></td></tr><tr><td>Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td><td>BD</td><td>297252</td><td></td></tr><tr><td>&lt;em&gt;Aspergillus fumigatous&lt;/em&gt; strain expressing green fluorescent protein</td><td></td><td></td><td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td></tr><tr><td>100 &amp;mu;m Cell strainers</td><td>BD Falcon</td><td>64753-00</td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Biological Safety Cabinet Class II</td><td></td><td></td><td></td></tr><tr><td>Sorval ST40 Centrifuge with swing bucket rotor</td><td></td><td></td><td></td></tr><tr><td>Leica DM1000 light microscope</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Cytospin 2 centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td>37 &amp;deg;C, 5%&amp;nbsp; CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>22 x 22 mm micro cover glass</td><td>VWR</td><td>48366-227</td><td>glass coverslips</td></tr><tr><td>6-well Cell culture plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>BDH0502-1LP</td><td></td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Leica TCS SP2 system with a laser point scanner</td><td></td><td></td><td>Mounted on DMIRE2 fluorescence microscope</td></tr></tbody></table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

肺是其中的宿主细胞经常接触到的微生物和微生物产品的接口。肺泡巨噬细胞是遇到吸入真菌和其它微生物的一线吞噬细胞。巨噬细胞和其他免疫细胞识别曲霉图案由病原体识别受体，并启动下游的炎症反应。吞噬细胞NADPH氧化酶产生活性氧中间体（投资回报）​​，是宿主防御的关键。虽然NADPH氧化酶是至关重要的中性粒细胞介导的宿主防御1 - 3，NADPH氧化酶的巨噬细胞中的重要性是不明确的。本研究的目标是界定在介导针对A.宿主防御NADPH氧化酶的巨噬细胞中的具体作用曲霉 。我们发现，在肺泡巨噬细胞NADPH氧化酶控制吞噬A的增长烟曲霉菌孢子4。在这里，我们描述了一种用于评估老鼠的能力lveolar巨噬细胞（AMS）来控制细胞吞噬黑曲霉的孢子（分生孢子）的生长。肺泡巨噬细胞进行染色体内并通过支气管肺泡灌洗（BAL）从小鼠十天后分离。巨噬细胞被镀到玻璃盖玻片上，然后种入绿色荧光蛋白（GFP）表达A.烟曲霉菌孢子。在指定的时间，将细胞固定和完好的巨噬细胞与吞噬孢子的数量通过共聚焦显微镜进行评估。

<html>从未经刺激的小鼠收集肺泡巨噬细胞通过BAL将导致基于细胞学一个纯度&gt; 95％的人口​​。在3和7小时（ 图2）的时间点之前的真菌生长的菌丝阶段，并允许对分生孢子的基因型之间的吞噬效率的比较。其中基因型可以是关于抑制吞噬的分生孢子于组织侵入菌丝阶段的过渡（ 图3）的能力进行比较，在14小时的时间点是。完好的巨噬细胞可以通过共聚焦显微镜的基础上...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

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

一种方法来评估分离的小鼠肺泡巨噬细胞，激光共聚焦显微镜，以控制吞噬曲霉菌孢子的生长的能力。

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 ...

assessing-anti-fungal-activity-isolated-alveolar-macrophages-confocal

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  Roswell Park Cancer Institute. 一种方法来评估分离的小鼠肺泡巨噬细胞，激光共聚焦显微镜，以控制吞噬曲霉菌孢子的生长的能力。 

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

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

科研

生物工程

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 ...

免疫与感染

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

研究肺泡巨噬细胞的作用乳腺癌转移

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

医学

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

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 ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

聚焦激光扫描显微镜为基础的定量分析 阿斯珀吉卢斯熏蒸 孔尼迪亚分布在全山光学清除鼠肺

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

巨噬细胞的真菌清除效率

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

作者聚焦:揭示木 霉基质 与哺乳动物免疫系统之间的相互作用

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

人外周血单核源性巨噬细胞内 基质木霉 分生孢子的活力测定

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

摘要

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此视频中的章节

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

活细胞视频显微镜病病原菌的吞噬作用

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞

研究肺泡巨噬细胞的作用乳腺癌转移

在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中

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

聚焦激光扫描显微镜为基础的定量分析阿斯珀吉卢斯熏蒸孔尼迪亚分布在全山光学清除鼠肺

人外周血单核源性巨噬细胞内基质木霉分生孢子的活力测定

人外周血单核源性巨噬细胞内基质木霉分生孢子的活力测定

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

摘要

探索更多视频

此视频中的章节

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

活细胞视频显微镜病病原菌的吞噬作用

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞

研究肺泡巨噬细胞的作用乳腺癌转移

在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中

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

聚焦激光扫描显微镜为基础的定量分析 阿斯珀吉卢斯熏蒸 孔尼迪亚分布在全山光学清除鼠肺

人外周血单核源性巨噬细胞内 基质木霉 分生孢子的活力测定

人外周血单核源性巨噬细胞内 基质木霉 分生孢子的活力测定

聚焦激光扫描显微镜为基础的定量分析阿斯珀吉卢斯熏蒸孔尼迪亚分布在全山光学清除鼠肺

人外周血单核源性巨噬细胞内基质木霉分生孢子的活力测定

人外周血单核源性巨噬细胞内基质木霉分生孢子的活力测定