医学真菌学真菌免疫学（SFB，JMB，JK，AW） - 这项工作已经由MRC和阿伯丁大学（MRC中心医学真菌学）和威康信托基金会战略奖支持。特别感谢的是从Chloe的资金支持。 TMH是由国立卫生研究院（NIH，代码RO1 093808）和纪念斯隆·凯特林癌症中心的资助是由美国国立卫生研究院授予P30 CA008748支撑。另外，TMH是传染病由宝来惠康基金支持的发病机制的调查。我们要感谢仔显微镜和组织学设施对他们的帮助和支持。

用于获得中性粒细胞和外周血单核细胞（PBMC）的协议是基于以前出版的方法9。使用来自健康志愿者的血液样本已通过阿伯丁大学的人类研究伦理委员会。开始之前，确保所有的伦理审批到位从健康志愿者和/或病人抽血所描述实验的目的。在冰上保持在可能情况下，以增加他们的生存细胞。 1. 烟曲霉文化条件<ol><li>如所描述的10制备葡萄糖基本琼脂培养基上。 <ol><li>调整媒体使用1M NaOH将pH为6.5并高压釜在120℃下20分钟。 </li><li>使其冷却至65℃。 </li><li>在无菌流罩的工作，倒入30毫升冷却介质成T75烧瓶中并允许与静止烧瓶的颈部冷却上的25毫升吸移管至create琼脂斜面。 </li></ol></li><li>条纹列于琼脂DSRED Af293- 烟曲霉菌株，在37℃+/- 5％CO 2孵育7天。两个烧瓶将在5mL的磷酸盐缓冲盐水（PBS）预生物素化，得到大约10 9个分生孢子。 </li></ol> 2. 烟曲霉标签和制备注意:当首次执行该测定中，制备亲本Af293- 烟曲霉菌株。采取两种菌株的样品中的微量离心管中并且如下所述仅标记每个菌株中的一个。这将允许一个有控制的分生孢子没有颜色和分生孢子用单一颜色，无论是红色荧光蛋白或AF633，测试设置和设备。 <ol><li>通过用30mL PBS + 0.05％吐温-80中浸渍培养收获烟曲霉分生孢子。通过40微米的细胞过滤到50mL管过滤所得悬浮液，以除去菌丝片段。 </li><li> Centrifuge在805×g离心10分钟，除去上清液，并在20毫升无菌PBS洗一次。在洗涤步骤中从相同菌株的两个板池分生孢子。 </li><li>在洗涤步骤之后，重新悬浮于5mL PBS的颗粒和转印分生孢子悬浮液的1ml等份到微量离心管中。 1mL的悬浮液各菌株的通常足以用于活细胞成像。包裹在箔和存储剩余的等分试样在4℃下。 </li><li>在9300×g下在室温下10分钟，并小心地分生孢子悬浮液的等分试样离心除去上清液。重悬沉淀分生孢子在1mL的0.05M的NaHCO 3 pH值8.3。 </li><li>通过在500μL的DMSO重构25毫克生物素制备10毫克生物素/ 200μL二甲亚砜（DMSO）储备液。添加10μL生物素/ DMSO原液到微量离心管中，在盖铝箔，并培育2小时上在4℃下的摇杆。 <ol><li>分装的生物素/ DMSO股票搜索解决方案的剩余部分n和在-20℃下，在随后的实验中使用的冻结。 </li></ol></li><li>离心10分钟，在9300×g下，小心地除去上清。重悬在1mL的100mM的Tris-HCl的pH 8.0的分生孢子沉淀1个小时以失活自由浮动生物素。 </li><li>离心10分钟，在9300×g下，小心地除去上清。用1mL无菌PBS洗涤沉淀两次，重悬沉淀在1mL的PBS中。 </li><li>溶解1毫克链霉亲和AF633的0.5毫升的PBS使一个2mg / mL的储备溶液。加入2 mg / mL的链霉亲和AF633的10μL每1mL分生孢子悬浮液的孵育在室温下在铝箔覆盖摇臂40分钟。其余的链霉亲和AF633可以等分被冻结在随后的实验中使用。 </li><li>离心10分钟，在9300×g下，小心地取出上清。重悬沉淀分生孢子在1mL的PBS中，并在1计数使用血球分生孢子:1,000稀释液（1:100，然后1:10）。调整分生孢子浓度3.6×10 6个 / mL的用CO 2独立媒体，在4℃下在箔和存储包裹。 注:现在的分生孢子都标有AF633，并将作为FLARE分生孢子被称为。 </li><li>可选:如果需要与肿分生孢子刺激，计数分生孢子（步骤2.9）后，稀释分生孢子至7.2×10 6个 / mL，在酵母氮基（YNB）培养基中，并在高压灭菌50加入500μL分生孢子悬浮液至4.5毫升YNB培养基姆·埃伦迈尔瓶中。覆盖并放置在烧瓶6小时振荡培养箱（37℃200rpm下）。 <ol><li>所述介质转移到15毫升管。离心机中以805×g于室温下10分钟，并在PBS中洗涤一次。再次离心，重悬沉淀在1毫升CO 2独立介质，给予3.6×10 6个分生孢子/ ml。 注:分生孢子经常聚集他们变得肿胀进行准确的计算困难后，建议用C来计算ounted数字静止使用分生孢子。 </li></ol></li></ol> 3.人体中性粒细胞和单核细胞的分离<ol><li>绘制20毫升的来自健康志愿者的静脉血液到含有乙二胺四乙酸（EDTA）2个10毫升血管中。用于分开每个供体，倒入20毫升血液中的50mL管中，用15mL PBS稀释。 </li><li>下垫的血液用15ml淋巴细胞分离溶液（密度1.077克/毫升）用注射器和针铁的。放置针在管的底部，轻轻迫使淋巴细胞分离溶液进行。离心机中以630×g离心20分钟，不制动和低加速。 </li><li>准备PBS，在冰上冷却。 注意:步骤3.4和3.5必须同时遵循以产生的单核细胞（3.4）和嗜中性粒细胞（3.5）。 </li><li>使用pastette，收获PBMC层。这是在黄色血清（上）和透明淋巴细胞分离溶液（低级）层之间的中心的层，和转移到新的50ml管中。 <ol><li>装满PBMC管高达50毫升用冷PBS和离心机中以582×g离心10分钟。去除上清，每20毫升的供体血液最初使用在1mL的PBS中用冷PBS重悬洗两次。 </li><li>使1:10稀释液通过将细胞悬浮液于160μLPBS中和台盼蓝的20μL的20μL计数。计数和血球细胞。 </li><li>通过加入26毫升热灭活的胎牛血清（FCS）和2.1毫升的0.5M的EDTA至500mL HBSS的制备缓冲溶液。离心将细胞在515×g离心10分钟，并再悬浮于每1×10 7细胞缓冲溶液40μL。 </li><li>细胞悬液转移到15毫升管中并每1×10 7细胞添加CD14微珠的10μL，拌匀，并在4℃下孵育15分钟，确保所述管混合，每5分钟。 </li><li>通过加入每1×10 7个细胞和CEN缓冲溶液1ml洗涤细胞trifuge在515×g离心10分钟。 </li><li>吸去上清液完全，重悬到1×10个8个细胞中的缓冲溶液500微升。 </li><li>根据制造商的说明放置每个样品1列上的磁分离器。首先用缓冲液500μL洗涤柱一次。 </li><li>通过该柱吸取细胞悬浮液的500μL，等待柱干燥，然后通过用缓冲溶液重复该三次洗涤。 </li><li>将新的15毫升管中的列的下面，并采取柱切断电磁分离器。然后冲洗细胞出列入用1mL的缓冲溶液的试管中。 </li><li>加入4毫升缓冲溶液和离心机在515×g离心10分钟，然后在1mL的Roswell Park Memorial研究所（RPMI）培养基重悬。 </li><li>使1:10稀释液通过将细胞悬浮液于160μLPBS中和台盼蓝的20μL的20μL计数。计数和细胞血球。 </li><li>调节细胞浓度为6×10 5 / mL的用RPMI和在基于玻璃35毫米成像菜种子200μL。在37℃用5％CO 2孵育过夜。 </li><li>第二天，在成像之前，取出RPMI并添加CO 2独立介质的200μL。 注意:这些单核细胞也可以分化成成像之前各种巨噬细胞子集。如果这是要探索的技术，一个很好的起点是由Ohradanova-Repic 等人最近的纸张。 1。 </li></ol></li><li>收获PBMC层后，除去所有的血清和大部分淋巴细胞分离溶液，但要小心，不要除去上冲粒料，因为这将包含大多数的嗜中性粒细胞的顶部上的小的白色带。 <ol><li>通过添加83克NH 4的Cl和在1升无菌水10克KHCO 3的制备10个的低渗裂解缓冲液贮液。保存在4°C。 </li><li>这个稀释10倍的股票用无菌水，并把它添加到管。然后，小心地反转3次，并在冰上孵育15分钟。 </li><li>离心机中以394×g离心10分钟，并再悬浮在低渗裂解缓冲液在冰中10分钟。 </li><li>离心机394 XG，用冷PBS洗两次。重悬在1mL每个供体的CO 2独立培养基。 </li><li>使1:10稀释液通过将细胞悬浮液于160μLPBS中和台盼蓝的20μL的20μL计数。计数与血球细胞。 </li><li>用于流式细胞术，调至浓度为6×10 5 / mL和400μL细胞悬浮液添加到24孔板中。对于显微镜，添加相同的细胞悬浮液的200μL到基于玻璃35毫米成像盘中。 注:中性粒细胞成像或流式细胞术应立即开始，由于他们的倾向，如果没有刺激发生凋亡。如果这是不可能的嗜中性粒细胞应保持在冰并尽快用作可能的（在同一天之内）。 </li></ol></li></ol> 4.流式细胞仪<ol><li>添加1.2×10 6 / mL的FLARE分生孢子200μL到在24孔板中的细胞中，然后添加20μg/ ml的伏立康唑的100μL。添加RPMI的300μL，并且在37℃用5％CO 2孵育16小时。 加入伏立康唑，防止孢子萌发:注意。 </li><li>添加25μL1 mM的卡尔科弗卢尔白色原液向每个孔为25μm的终浓度，并在37℃用5％CO 2孵育10分钟。 </li><li>离心机中以582×g离心10分钟。除去上清液，并用PBS洗两次。 </li><li>收获贴壁细胞（单核细胞）中，用3mM的EDTA添加1毫升的PBS至每个孔，并在37℃用5％CO 2孵育10分钟。轻轻用移液管洗涤细胞从板上收集的细胞在管中。 </li><li>在FACS缓冲液洗一次（2％胎牛血清到PBS），然后在FACS缓冲液悬浮于FACS分析。 <ol><li>对于FACS分析，首先流式细胞仪调节流量的补偿参数，并设置使用单彩色补偿管正面门，包括分生孢子没有颜色:红色荧光蛋白+分生孢子，AF633 +分生孢子，并卡尔科弗卢尔白+分生孢子（如在4.2生成）。 </li><li>设置免费分生孢子门上了基于使用没有颜色的分生孢子侧向散射。通过排除免费分生孢子门上的前向和侧向散射设置单元门。 </li><li>通过记录10,000个事件分析样品管中。 注:用活分生孢子相关细胞将红色荧光蛋白+和AF633 +。与死亡有关的分生孢子细胞只会AF633 +。那些没有从事分生孢子会dsRed-和AF633-旁观者细胞。分生孢子，从事细胞群进一步分析了太平洋上的蓝色通道卡尔科弗卢尔白染色。已经内化分生孢子仍然存在细胞外将卡尔科弗卢尔白+，和分生孢子会卡尔科弗卢尔白色 - 。 </li></ol></li></ol> 5.活细胞显微视频注意:显微镜的选择将取决于什么是本地可用的，但在显微镜的设置将需要包括倒置的阶段，加热至37℃和适当的激发/发射过滤器的DsRed（五百六十一分之五百三十二nm）与AF633环境室（633纳米）。 FLARE分生孢子不与488 nm激光代替五百六十一分之五百三十二nm激光对红色荧光蛋白的工作。当与此第一次执行该实验中，使用控制分生孢子没有颜色和单色测试对于背景荧光和红色荧光蛋白和AF633信道之间的渗出通过。 <ol><li>打开之前的实验显微镜加热器，并允许足够的时间使环境控制腔室温至37℃。以稳定采取的腔室温度的时间将不同的显微镜设置而变化。 </li><li>打开显微镜和计算机，LO广告中的成像软件。安装在显微镜载物台的摄像培养皿中并调节焦距以找到人类嗜中性粒细胞或单核细胞。 </li><li>对于红色荧光蛋白和AF633，在采集设置设定的激光功率到10％和曝光时间为1秒。 </li><li>取出成像滑动，并添加100微升的3×10 6个 / mL的FLARE分生孢子悬浮液到适当的孔中（总体积300μL/孔）。录制FLARE分生孢子添加到菜的时间。 </li><li>返回滑动到舞台和调整的DsRed和AF633摄像机的灵敏度清楚地看到分生孢子但不是过度曝光图像。如果需要多点采集设置一个阶段点列表。 </li><li>开始成像时的所有点都在焦点中，通道被优化和循环时间和持续时间被设置。周期时间和成像持续时间取决于试验问题。的目标是保持周期时间尽可能地短（≤2分钟）1分钟在UPT的深入分析允许AKE动态。 </li></ol>

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Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes.</a> J Clin Invest. 69 (3), 617-631 (1982).</li></ol>

作者什么都没有透露。

该方法描述了一种新颖的体外方法，研究对烟曲霉的人原代巨噬细胞使用荧光曲霉记者（FLARE）分生孢子，活细胞成像的抗真菌活性和流式细胞术。先前的研究已经证明了无论是在鼠实验真菌感染体内 和抗真菌免疫6,7的体外评估使用FLARE分生孢子的附加值。组合FLARE分生孢子与此下一代活细胞成像技术允许一个在安装过程中在体外动态相互作用以测量单个分生孢子生存能力。 所描述的方法来研究它们的抗真菌反应的免疫细胞的某些细胞过程的具体作用和重要性的潜力。此外，从患特定患者吞噬细胞的抗真菌反应在他们的免疫系统中定义的单个组件的缺陷可以更详细地评估。很早就和初始抗真菌响应可以非常详细地在连续成像的6小时进行研究。这允许待检测甚至细微的差别，如巨噬细胞迁移的朝向真菌的速度，内化速率，杀真菌事件12，这将使用常规方法的吞噬作用是无法区分的动力学。 任何细胞类型都可以在本方法中使用;选择在此使用的细胞类型，因为这些吞噬细胞构成防御在人呼吸道的第一和第二线对烟曲霉 13。有趣的是，可以单核细胞之间和中性粒细胞是如何与休息和肿分生孢子和菌丝接合可以观察到非常清晰的差异。单核细胞几乎没有迁移到分生孢子，而嗜中性粒细胞显示更多的迁徙活动。附加荧光三月KERS如免疫细胞活死标记可以被包括在试验提供见解以下曲霉相互作用吞噬细胞的生存。该技术有趣潜在的未来应用包括不同的宿主细胞类型的共培养物中，抗真菌反应的后果（ 例如 ，在上皮单层，单核细胞衍生的树突状细胞与嗜中性粒细胞一起巨噬细胞），和反应性的实时测量以下用曲霉刺激氧的产生或嗜中性粒细胞的胞外陷阱的形成。 有几点需要注意使用该协议，最重要的是需要在实验室设置:具有倒置阶段的显微镜，环境室稳定在37℃，激发/发射过滤器的DsRed（五百六十一分之五百三十二nm）与AF633（ 633纳米）。显微镜，以保持细胞在聚焦和保留油浸（米时特别相关的能力ULTI点采集）应该被周期性地监测由于成像的持续时间长。对于杀菌活性的定量流式细胞仪设备是必需的。此外，适当的体制和伦理审批需要在地方与健康供者和患者的血液样本和基因操纵真菌工作。强烈建议使用新鲜的血液样本（在同一天使用），增加细胞活力和维护功能。理想情况下，细胞的分离被绘制血样后立即进行和嗜中性粒细胞分离后立即成像。 总之，针对烟曲霉中描述了一种下一代活细胞成像技术来非常详细吞噬细胞的抗真菌活性评估。这项技术可以在对黑早期先天免疫防御提供了一个独特的见解单个细胞真菌的相互作用。

烟曲霉是一种条件真菌病原体，并在免疫受损宿主1,2侵袭性肺感染的最常见的真菌病因。理解宿主免疫细胞如何识别和消除黑是真菌研究的一个重要领域，然而，当前可用的杀菌试验有显著的局限性。测量的杀真菌活性的常用的方法包括比色测定和菌落形成单位3,4判定。然而，这些方法在一个单一的时间点，而不是观看主机和病原体之间的动态相互作用提供对真菌存活的信息。因此，真菌是否已被杀害或简单（临时）限制在生长或代谢活性，这些方法都没有考虑到。我们已经开发出能够观测fungici的方法直接DAL活性，同时同时捕获关于吞噬作用，吞噬细胞迁移和真菌生长的抑制的详细信息。 先前，协议，使用实时成像如由小鼠巨噬细胞系5以测量白色念珠菌的吞噬作用的装置发布。这个概念现在已经开发了进一步利用荧光记者曲霉 （FLARE）分生孢子6，7，8，以解决我们的免疫细胞曲霉相互作用的了解的知识差距。这些分生孢子表达红色荧光团（dsRed）组成的和标记有第二荧光团（Alexa氟633）。一旦FLARE分生孢子被杀害，它将停止生产DSRED，其余DSRED变淡，而AF633保持荧光并将其绑定到分生孢子。这将创建与活的和死夫有着明显的区别期间活细胞成像的细胞的NGAL和流式细胞术，并能够以下它们与免疫细胞相互作用个体分生孢子的命运的跟踪。 所描述的测定法提供可视化宿主免疫细胞和曲霉属之间的相互作用的有效方法，并且将在解开负责天然免疫细胞的抗真菌效应物机制的细胞信号传导途径是相当大的价值。其它应用包括可视化变化曲霉生长，如从静息到肿分生孢子，或从肿分生孢子到菌丝开关，如何影响吞噬细胞识别和功能。 以下方案详细描述了如何获得人单核细胞和嗜中性粒如何准备FLARE分生孢子;如何捕捉他们的回答与视频显微镜和流式细胞仪。虽然使用捐助血液中分离人体免疫细胞的在这里，这说明技术已使用各种鼠细胞群的证明同样有效。

<table><tbody><tr><td>Glycerol</td><td>Sigma</td><td>G6279</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>T75 flask</td><td>Greiner Bio-One</td><td>658 175</td><td>http://www.greinerbioone.com/</td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-019</td><td>https://www.thermofisher.com/</td></tr><tr><td>Tween-80</td><td>Fisher Scientific</td><td>10592955</td><td>https://nl.fishersci.com/</td></tr><tr><td>40 &amp;micro;m filter</td><td>Thermo Fisher Scientific</td><td>22363547</td><td>https://www.thermofisher.com/</td></tr><tr><td>15 mL Falcon tube</td><td>Greiner Bio-One</td><td>188 261</td><td>http://www.greinerbioone.com/</td></tr><tr><td>50 mL Falcon tube</td><td>Greiner Bio-One</td><td>227 261</td><td>http://www.greinerbioone.com/</td></tr><tr><td>MilliQ</td><td>Millipore</td><td>QGARD00R1</td><td>Nanopure water works similarly. http://www.merckmillipore.com/</td></tr><tr><td>Biotin</td><td>Molecular Probes</td><td>B-6352</td><td>https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D5879</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>Streptavidin-AF633</td><td>Molecular Probes&amp;nbsp;</td><td>S-21375</td><td>AF633 is the only tested fluorophore so far that remains visible in phagosomes. https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html</td></tr><tr><td>EDTA blood tubes</td><td>Greiner Bio-One</td><td>455036</td><td>Any blood tubes with an anti-coagulating agent should work. http://www.greinerbioone.com/en/start/</td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-1440-03</td><td>http://www3.gehealthcare.com/</td></tr><tr><td>Trypan blue</td><td>Thermo Fisher Scientific</td><td>SV3008401</td><td>https://www.thermofisher.com/</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14170112&amp;nbsp;</td><td>https://www.thermofisher.com/uk/en/home/brands/gibco.html</td></tr><tr><td>CD14 microbeads</td><td>Myltenyi Biotec</td><td>130-050-201</td><td>Other monocyte isolation methods can be used as well, we prefer this method as it gives a consistent high purity of monocytes without being labor intensive. http://www.miltenyibiotec.com/en/</td></tr><tr><td>MS Columns</td><td>Myltenyi Biotec</td><td>130-042-201</td><td>See comment at CD14 microbeads. http://www.miltenyibiotec.com/en/</td></tr><tr><td>RPMI + Glutamax</td><td>Gibco</td><td>72400-021</td><td>https://www.thermofisher.com/uk/en/home/brands/gibco.html</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; independent medium</td><td>Thermo Fisher Scientific</td><td>18045054</td><td>Necessary if there is not sufficient CO&lt;sub&gt;2&lt;/sub&gt; exchange during imaging. https://www.thermofisher.com/uk/en/home</td></tr><tr><td>&amp;micro;-slide 8 well glass bottom</td><td>Ibidi</td><td>80827</td><td>This is the imaging dish that we use, however any glass imaging dish of proper size should work. http://ibidi.com/</td></tr><tr><td>UltraVIEW VoX 3D Live Cell Imaging System</td><td>Perkin Elmer</td><td>L7267000</td><td>Includes volocity software for acquisition and analysis. http://www.perkinelmer.com/</td></tr><tr><td>Calcofluor white</td><td>Sigma</td><td>18909</td><td>http://www.sigmaaldrich.com/</td></tr><tr><td>Voriconazole</td><td>Sigma</td><td>PZ0005</td><td>http://www.sigmaaldrich.com/</td></tr><tr><td>BD LSRII</td><td>BD Biosciences</td><td></td><td>Flowcytometer used. https://www.bdbiosciences.com/</td></tr></tbody></table>

live imaging of antifungal activity by human primary neutrophils and monocytes in response to a. fumigatus

烟曲霉是一种条件真菌病原体引起免疫妥协的宿主侵入性感染具有高的病死率。研究调查对烟曲霉免疫反应已经缺乏体外测定特异性免疫细胞的抗真菌活性一致和可靠的试验的限制。描述了一种新的方法来评估主单核细胞和从对烟曲霉的人供体使用荧光曲霉记者（FLARE）分生孢子嗜中性粒细胞的抗真菌活性。这些分生孢子含有遗传编码的DsRed记者，其组成由活FLARE分生孢子表达，并用Alexa氟633，这是吞噬溶酶体内降解具有抗性，从而使之间存活和死亡的烟曲霉分生孢子区别是外部标记。视频显微镜，流式细胞仪随后被用于可视化互动分生孢子和先天免疫细胞之间的离子，评估杀真菌活性，同时还对吞噬细胞迁移，吞噬作用和真菌生长的抑制提供了丰富的信息。这种新颖的技术已经提供了令人兴奋的新见解对烟曲霉的主要免疫细胞的宿主-病原体相互作用。要注意执行该法，包括必要的显微镜和流式细胞仪设备所需的实验室设置，并与人供体的血液和基因操纵真菌工作能力是很重要的。然而，该测定能够产生大量的数据，并能揭示详细见解抗真菌响应。该协议已经被成功地用于研究针对烟曲霉初级免疫细胞的宿主-病原体的相互作用。 要注意进行这一测定所需的实验室设置，包括必要的显微镜和流式细胞仪F IT是非常重要的教学设施，并有能力与人类供体血液和遗传操作的真菌工作。然而，该测定能够产生大量的数据，并能揭示详细见解抗真菌响应。该协议已经被成功地用于研究针对烟曲霉初级免疫细胞的宿主-病原体的相互作用。

代表性的结果示下面所描述的协议。 图1示出与人类嗜中性粒细胞和FLARE分生孢子的代表性6小时的活细胞视频。红色荧光蛋白（红色）是由分生孢子自己，而他们已经用AF633（品红）表示。 图2是从从类似电影的单个时间点的图像作为显示在图1中 ，示出了间活的和死的FLARE分生孢子的差异。死分生孢子由失去他们的红色荧光蛋白（红色）信号，同时保持明亮AF633（品红色）的荧光从活分生孢子区分。 烟曲霉分生孢子经常吞噬超过6小时内生存。因此，为了有效杀死量化，流式细胞术示出了用于在图3中16小时温育期图。用一个肺泡巨噬细胞CE获得该数据LL线作为一个例子，但可以使用任何细胞类型中进行。 图4中的迁移显示在刺激人中性粒细胞，以及它们的速度和方向运动的第一个小时。 图5示出已摄取1个，2个，3个或更多个分生孢子而图6显示了萌发成菌丝分生孢子的数目嗜中性粒细胞和单核细胞的百分比。 <img alt="图1" src="/files/ftp_upload/55444/55444fig1.jpg" /> 图1:人嗜中性粒细胞和FLARE分生孢子的活细胞视频显微镜电影。嗜中性粒细胞从人血液中分离，并与在CO 2独立介质FLARE分生孢子以1:1的比率接种一起:3。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在一段6小时来捕获。比例尺=10μm以下。甲在视频放大示出了具有分离的，以澄清在左上角与DIC的FLARE分生孢子的作用通道，红色荧光蛋白（红色）在右上角，AF633（绿色）在左下方和右下方的合并的视频。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/Fig_1.avi">请点击这里下载该视频。</a> <img alt="图2" src="/files/ftp_upload/55444/55444fig2.jpg" /> 图2:单一时间点图像展示之间活的和死FLARE分生孢子的差异。人中性粒细胞用FLARE分生孢子刺激，并用旋转盘共聚焦显微镜成像。图像从生成的电影拍摄。同时保持其AF633荧光（绿色:左下）信号:死孢子从菌丝由已经失去了他们的红色荧光蛋白（右上红）区别开来。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig2large.jpg">请点击此处</a><html>查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55444/55444fig3.jpg" /> 图3:量化与吞噬和烟曲霉分生孢子杀害验证。鼠肺泡巨噬细胞（MH-S）在一个1 FLARE分生孢子孵育:1的比例在伏立康唑的存在下16个小时。与活分生孢子相关MH-S细胞中示出了红色栅极（DsRed的+ AF633 +）。与死分生孢子相关MH-S细胞中示出了蓝色栅极（DsRed的-AF633 +）。旁观者MH-S细胞中示出了金栅极。卡尔科弗卢尔白，结合真菌细胞壁的细胞不可渗透的荧光染料，用于区分细胞外分生孢子从细胞内的分生孢子（卡尔科弗卢尔白+）（卡尔科弗卢尔白色 - ）。作为一个额外的验证的方法，它被示为具有2μM细胞松弛素D中治疗抑制分生孢子摄取MH-S细胞。e.jove.com/files/ftp_upload/55444/55444fig3large.jpg&quot;&gt;Please点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55444/55444fig4.jpg" /> 图4:人嗜中性白细胞和抗烟曲霉的分生孢子的单核细胞迁移。嗜中性粒细胞从人血液中分离，并与在CO 2独立介质FLARE分生孢子以1:1的比率接种一起:3。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在6小时的时间捕获。每场的所有单独的人中性粒细胞迁移的手动跟踪的使用跟踪软件对静息（A）和肿胀（B）分生孢子1个小时。数据表示为相同的供体的各条件1帧单元。曲折因子（C），的directio的量度纳尔运动和速度（D）然后，使用相同的软件进行定量。平均值±SEM对被示出具有每个供体30个细胞在2个独立实验3个供体。 *:P &lt;0.05（韦尔奇校正t检验）。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig4large.jpg">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55444/55444fig5.jpg" /> 图5:由人嗜中性白细胞和单核细胞，烟曲霉的分生孢子的吞噬作用。分别为3，:嗜中性粒细胞和单核细胞是从人血液中分离，并与在CO 2独立介质FLARE分生孢子接种在一起的比例为1。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在一段6小时来捕获。吞噬作用测定为细胞含有的量刺激4个小时后荷兰国际集团FLARE分生孢子。数据被呈现为平均值±SEM从3个供体和在2个独立实验每个供体3帧。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig5large.jpg">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55444/55444fig6.jpg" /> 图6: 由嗜中性粒细胞和单核细胞 ，烟曲霉 分生孢子发芽的抑制作用 。分别为3，:嗜中性粒细胞和单核细胞是从人血液中分离，并与在CO 2独立介质FLARE分生孢子接种在一起的比例为1。成像是在37℃下直接发起了与一旋转盘共聚焦显微镜。图像是在1分钟和55秒的间隔在6小时的时间捕获。真菌萌发的抑制是通过测量分生孢子是有细菌的百分比检查后共培养2,4和6小时inated。数据被呈现为平均值±SEM从3个供体和在2个独立实验每个供体3帧。 <a href="http://ecsource.jove.com/files/ftp_upload/55444/55444fig6large.jpg">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus at JoVE.com

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

在这里，我们描述了一种协议与活细胞视频显微结合使用荧光曲霉分生孢子记者评估初级人类免疫细胞的抗真菌活性在实时和流式细胞术。所生成的数据提供深入了解宿主细胞曲霉相互作用如杀真菌活性，吞噬作用，细胞迁移和真菌生长的抑制。

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 ...

live-imaging-antifungal-activity-human-primary-neutrophils-monocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

10.1K 次观看.  University of Aberdeen. 在这里，我们描述了一种协议与活细胞视频显微结合使用荧光曲霉分生孢子记者评估初级人类免疫细胞的抗真菌活性在实时和流式细胞术。所生成的数据提供深入了解宿主细胞曲霉相互作用如杀真菌活性，吞噬作用，细胞迁移和真菌生长的抑制。 

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

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

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

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 ...

科研

免疫与感染

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 ...

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 ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

实验性曲霉病肺真菌负担的组织学定量测定

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

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 ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

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 ...

用于筛选不同信号通路的中性粒细胞功能检测

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

作者聚焦:使用 NeutroFun 筛选方案平衡中性粒细胞功能评估的速度、成本和准确性 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

一套快速了解中性粒细胞功能的筛查技术

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

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

摘要

探索更多视频

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

活细胞视频显微镜病病原菌的吞噬作用

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

实验性曲霉病肺真菌负担的组织学定量测定

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

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用

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

一套快速了解中性粒细胞功能的筛查技术

一套快速了解中性粒细胞功能的筛查技术

一套快速了解中性粒细胞功能的筛查技术

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

摘要

探索更多视频

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

活细胞视频显微镜病病原菌的吞噬作用

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

实验性曲霉病肺真菌负担的组织学定量测定

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

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

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

一套快速了解中性粒细胞功能的筛查技术

一套快速了解中性粒细胞功能的筛查技术

一套快速了解中性粒细胞功能的筛查技术

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用

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