Name: visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy
Uploaded: 2014-10-21T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

这项研究是由美国国立卫生研究院奖5T32AI07506，5R01AI033774，5R37AI033142，5R01AI052733部分支持。

所有的动物工作是按照规定和研究所动物研究在医学阿尔伯特·爱因斯坦医学院的指导方针进行。 C的1生长条件新生隐球菌 H99（血清型A） <ol><li>个人成长三于沙氏琼脂平板隐球菌 （Cn）的殖民地。 </li><li>在实验前一天，选择一个菌落，接种10毫升沙氏肉汤。 </li><li>让文化在37℃生长过夜晃动在250转的速度。 </li></ol> 2，分离和C57准备原发性骨巨噬细胞/ BL6小鼠<ol><li>通过将动物在CO 2室中，直到不再呼吸安乐死的小鼠。作为第二个措施，与颈部脱臼开始解剖前的脖子。消毒的整个身体用70％的乙醇。 </li><li>在无菌的DMEM取出两个股骨和胫骨和骨的地方。 </li><li>除去使用去多余的组织和肌肉licate擦拭，直到骨头是完全干净的。 </li><li>将唯一的完整的骨头含有70％乙醇的培养皿中孵育3分钟，以杀灭微生物相关。在培养皿中，用无菌DMEM培养基中取出的骨头和地点。 </li><li>经过精心切割的骨端。保持骨骼在一个空的50ml锥形管置于冰上，小心冲洗使用将25g针头穿过每个骨10毫升冷的DMEM。 注:每个鼠标将产生4骨头（2股骨，胫骨2）。因此，一个鼠标应该产生大约40 ml的细胞悬液。 </li><li>离心细胞悬浮液在室温下以650×g的10分钟。 </li><li>在该离心分离，制备和过滤消毒骨髓巨噬细胞饲养介质其由DMEM补充有20％的L929，10％胎牛血清，10％NCTC-109，1％青霉素 - 链霉素，1％HEPES，1％L-谷氨酰胺的，1％非必需氨基酸，0.1％2 - 巯基乙醇。 </li><li>悬浮产生的沉淀用10毫升喂媒体。通过细胞suspens离子通过一个70微米的细胞过滤破坏细胞团块，并删除大量的碎片。 </li><li>板1 ml的细胞悬浮液到10ml送入介质的培养皿中被用于组织培养用（共10片）中指定。 </li><li>让细胞在37℃下附着有10％的CO 2。加入5毫升的新鲜进料的介质在第3天第7天，完全取代馈送媒体和巨噬细胞准备好被使用。 </li><li>在实验前一天，从分离板的巨噬细胞去除送入介质，并添加一个温柔的细胞剥离试剂板5毫升。 </li><li>孵育5-10分钟，在37℃，并除去巨噬细胞与温和移液。 </li><li>离心沉淀细胞，在650×g离心10分钟。 </li><li>悬浮颗粒在1毫升饲养媒体。用1:20稀释，用移液管将10μl细胞悬浮液到血球计计算的巨噬细胞的浓度。 </li><li>使用14个毫米的玻璃筑底培养皿，板甲浓度为1×10 5的巨噬细胞，直接在其上拥有最大200微升的内部不错，所以采取谨慎不超过这个量， 如图4。 </li><li>使细胞通过将餐具在37℃培养箱中培养1小时，坚持以玻璃培养皿。 </li><li>加1-2喂养补充有LPS的介质的混合物在1毫克/毫升和IFNG，在500单位/毫升，并让细胞孵育过夜。 </li></ol> 3，联合培养初级小鼠巨噬细胞和C新型隐球菌 <ol><li>对实验的当天，除去1毫升过夜接种培养和沉淀的酵母细胞通过离心5分钟，在420×g下。 </li><li>用无菌PBS洗涤细胞3次，以去除介质和细胞外隐球菌产品，如多糖glucuronoxylomannan（GXM）的速度和时间上面提到的。 </li><li>重悬细胞沉淀在1ml无菌PBS中，并以1:100稀释。移取10微升的t他稀释到血球计数和细胞。添加酵母细胞以1 MOI:5。例如，如果有1×10 5个巨噬细胞/孔，使道道的5×10 6个/ ml的细胞悬浮液，并添加100微升的该溶液为5×10 5的C n的总量。 </li><li>添加的隐球菌细胞悬浮液算出的体积为1毫升的单克隆抗体18B7抗体送入介质中的10微克/毫升的浓度。孵育细胞悬浮液在室温下搅拌5分钟。 </li><li>从玻璃中取出送入介质见底培养皿洗1X用无菌PBS。添加调理道道100微升直接向好。 </li><li>使细胞温育2小时，在37℃，用10％的CO 2。 </li><li>经过共同培养，通过显微镜检查的巨噬细胞已经内化道道。被认为是内化，新型隐球菌细胞应该清楚地巨噬细胞的范围内看到。 </li><li>洗涤培养皿3次，用无菌PBS洗涤以除去任何和所有胞外Ř道道。 </li><li>加1-2毫升不添加单克隆抗体18B7喂养媒体，并成立了显微镜。 </li></ol> 4，非可视化的溶胞吐作用的实时利用数字光学显微镜注:要执行这些实验中，自带的，包括二氧化碳输送和加热装置，需要一个附加的孵化室的显微镜。 <ol><li>在实验前，预先温热的孵育室中至少30分钟至37℃。确保CO 2单元在实验开始时读取的5％。 </li><li>发生玻璃见底培养皿上的显微镜平台，涵盖与CO 2盖，并关闭所有的门，以确保无热逃逸。 </li><li>使用10倍的目标，在相差显微镜，专注于受感染的巨噬细胞的清场。基于该实验中，确定所使用的多少的巨噬细胞需要研究的目标。 </li><li>成立显微镜软件TA磕的图像，每4分钟一24小时周期。 </li><li> 24小时后，实验将达到完工，共361图像将被收集。截至5％的压缩.jpegs导出这些图像。使用的Image J软件，查看图像的视觉堆叠在每秒5帧。如果影片需要查看的出版物或演示文稿，将其保存为要么是avi或。根据mov文件上的格式最适合的浏览器。 </li></ol>

<ol>
	<li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+Interactions+With+the+Host+Immune+System.">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+interactions+with+macrophages:+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen.">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li><li>Sabiiti, W., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans.">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li><li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pathogenic+bacteria+and+fungi+&#8212;+a+case+of+convergent+evolution.">Intracellular pathogenic bacteria and fungi &#8212; a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages.">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16 (2007).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell-to-cell+spread+of+a+pathogenic+yeast.">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15 (2007).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages.">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expulsion+of+live+pathogenic+yeast+by+macrophages.">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2/3+complex-mediated+actin+polymerisation.">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li><li>Carnell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis.">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li><li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans.">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li><li>Bain, M. J., Lewis, E. L., Okai, B., Quinn, J., Gow, A. R. N., Erwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal genetics and biology. 49 (9), 677-678 (2012).</li></ol>

作者宣称，我们没有竞争的经济利益或其他利益冲突。

在这里，我们描述了其中的真菌细胞巨噬细胞的相互作用可以被记录并实时在24小时内进行分析的方法。我们的实验室已使用这个协议来研究许多非裂解性胞吐作用的时间方面，将继续使用实时显微术以确定围绕该过程的形态的组件。 这种技术产生理想的效果，特别应注意在协议的某些步骤。之前被镀夜间细胞密度是重要的，因为太多的细胞可以创建的电影是不可用作运动和各个细胞的相互作用的视野被阻碍，而过少的细胞可以得到的数据，是不是显著。的目标是实现巨噬细胞，使得个体的巨噬细胞可随后用于感染周期的持续时间的间隔均匀场。在此同样，洗去细胞外道道是关键一步，因为前胞道道通将分随着时间的推移这可能掩盖了现场，并导致巨噬细胞的知名度损失。根据动画的长度，帧可以移出焦点，这使得图像不可用，研究，因此，在记录需要在各个点的感染过程中进行检查的。 如所述，有各种方法这一技术可以被修改以适应其它实验参数范围内。许多不同的荧光染料和标记可用于突出膜和吞噬体动力学，但应该谨慎光漂白的和影响，这可能对细胞的健康。有多种类型的细胞也可以使用，如在与其他真菌菌株一起J774.1巨噬细胞样细胞，单核细胞或变形虫，研究可能出现的差异。我们建议使用10X的目标，因为我们的实验需要大量的细胞的采集数据，但是人们可以用更高的客观的研究只是一个几个细胞。每一个拍摄图像的定时也可以被调整。以图像每4分钟24小时产生一个运行大约1分钟长时，所有帧被压缩电影。增加任一方面（感染的持续时间或帧之间的时间）的增加可能是一个问题，一些实验室的需要存储的文件的数量。 非裂解性胞吐作用是，在感染过程中迅速地并在不同的时间间隔中出现令人难以置信的动态过程。因此，试图捕获的巨噬细胞进行使用其它方法，如传输和扫描电子显微镜这种相互作用可以是极其罕见的。由于如何样品固定和处理的性质，人们不能确定什么被认为是决定性的非裂解性胞吐作用与其他细胞过程如溶解或凋亡。此外，非溶胞胞吐的一大特点是，宿主与病原体通过保持<html> BLE事后这恐怕是不能从固定的影像推断，因为这些方法只能在某一时刻产生过程的快照。 而这个协议可提供细胞过程的无数的图像，也有在使用这种技术来研究宿主病原体相互作用的一些局限性。低目标，我们使用来研究了大量的巨噬细胞在同一时间不允许足够高的分辨率的图像进行可视化的吞噬体和细胞膜，这不​​是非裂解性胞吐作用的很好的理解方面之间的相互作用。一旦所有图像已被记录，下面的许多巨噬细胞的事后分析可能是非常烦琐和耗时的。巨噬细胞，甚至那些附着于玻璃，可以非常游动，并会移进和移出该帧的速度非常快。这可以使它很难跟随个体的巨噬细胞从开始到结束，特别是那些接近所述框架的周边。 jove_content"&gt;尽管有这些限制，这是协议可以以多种方式来研究的宿主 - 病原体相互作用的许多不同的方面可以使用。

隐球菌细胞和巨噬细胞之间的真菌感染的阶段有据可查1-3。后的酵母细胞被巨噬细胞摄取，各种相互作用，可能会发生:在巨噬细胞可裂解释放其真菌负载到细胞外空间，或者它可以通过其细胞膜4的范围内保持酵母细胞控制感染。非裂解性胞吐作用，一种方法，其中巨噬细胞中擦去一些隐球菌细胞或所有进入周围环境或邻近细胞和宿主和病原体保持存活5:然而，几年前，一个新的结果是由两个独立组中所述-8。一些研究试图了解这种相互作用背后的分子机制，但我们仍然没有十足的把握是什么推动了这一现象。 的能力来捕获实时图像以及随后分析多个侵染编巨噬细胞允许一个回答有关期间真菌感染周围的形态，甚至压力巨噬细胞的关于时间，空间能力，改变非溶胞胞吐作用的物理性质的许多问题。通过使细胞被安置在一个环境是相媲美的一个孵化器，我们就可以一睹巨噬细胞真菌细胞相互作用的动态特性。研究人员已用这种技术来研究该方法的各个组成部分。在此过程中，吞噬体中的作用，用荧光染色和肌动蛋白封闭剂9变得显而易见，而另一组用这种方法来显示，非裂解性胞吐作用被阻断在已经有WASH核蛋白质结构域删除的10个细胞。细胞因子信号传导的效果也已经使用实时显微镜11确定。这一过程并不限定于C。新型隐球菌 ，因为它也被观察到与白色念珠菌，anotheř病原真菌有感染的巨噬细胞12的能力。 这些研究结果说明该方法可以提供丰富的信息，我们所知甚少的领域的例子。

<table><tbody><tr><td>Sabouraud Dextrose Agar</td><td>BD</td><td>DF0109-17-1</td><td></td></tr><tr><td>Sabouraud Dextrose Broth</td><td>BD</td><td>DF0382-17-9</td><td></td></tr><tr><td>Dubelcco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Corning Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Atlanta Biologicals</td><td>S12450</td><td></td></tr><tr><td>NCTC 109</td><td>Invitrogen</td><td>21340-039</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>HEPES Buffer</td><td>Corning Cellgro</td><td>25-060-Cl</td><td></td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Corning Cellgro</td><td>25-025-Cl</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Invitrogen</td><td>21985-023</td><td>Toxic</td></tr><tr><td>3003 Tissue Culture Petri Dishes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>08772E</td><td></td></tr><tr><td>CellStripper</td><td>Corning Cellgro</td><td>25-056-Cl</td><td></td></tr><tr><td>Mat-tek Glass Bottom Culture Dishes</td><td>MatTek</td><td>P35GC-1.5-14-C</td><td></td></tr><tr><td>LPS</td><td>Sigma</td><td>L3137</td><td></td></tr><tr><td>Mouse IFN-g</td><td>Roche</td><td>NC 9222016</td><td></td></tr><tr><td>mAb 18B7</td><td>Non-commerical antibody produced in our lab</td><td></td><td></td></tr><tr><td>Axiovert 200M Microscope with Incubating Chamber</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

巨噬细胞中由新型隐球菌感染的许多方面进行了广泛研究，并很好的定义。然而，尚不清楚一个特定的交互作用是非裂解的胞吐作用。在这个过程中，酵母细胞是由知之甚少机构留下两个巨噬细胞和道道可行释放到细胞外空间。在这里，我们介绍了如何遵循了大量单独感染的巨噬细胞通过时间已过显微镜24小时感染期。受感染的巨噬细胞被容纳在加热室中以在CO 2气氛中附连到显微镜，它提供了相同的条件下的细胞培养孵化器。在线数码显微镜可以提供一台主机和病原体不能从静态图像之间的动态交互的信息。能够想象每个感染细胞可以提供线索，巨噬细胞是如何处理的真菌感染，反之亦然。这种技术ìSA强大的工具，学习，后面的一个复杂的现象的动态。

该技术生产出的图像可以以多种方式来收集周围的细胞有吞噬C.后会发生什么情况的信息进行分析， 新型隐球菌，正如在图1中 ，大约有100噬细胞是任何受感染或未受感染。这些巨噬细胞中的每一个给观众来研究一个非常微观层面宿主 - 病原体相互作用的机会。如图2和图3示出，也有能够在巨噬细胞和酵母细胞之间发生不同的结果，并且甚至介于巨噬细胞-巨噬细胞。巨噬细胞可被评价为处于非裂解性胞吐作用，细胞 - 细胞传递，或可能发生的任何其他细胞过程。此类事件的感染过程中的定时，也可以根据当电影被记录，所设置的参数确定。 <img alt="图1" fo:content-width="5英寸"SRC ="/文件/ ftp_upload / 52084 / 52084fig1highres.jpg"WIDTH ="500"/&gt; 图1:感染的巨噬细胞的领域本图显示了单独感染的巨噬细胞的整个领域，在24小时的感染期的开始。但是应当注意的是，该字段是自由胞外的C n的和更重要的是，它是如何明确其巨噬细胞被感染（由大箭头所指示的）和含有无真菌细胞（由小箭头所示）。比例尺条为50微米。 <a href="https://www.jove.com/files/ftp_upload/52084/Updated_Field_of_Macrophages.mov" target="_blank">短片1</a> :巨噬细胞C的可视化新型隐球菌 24小时感染期，影片的初始帧显示，许多巨噬细胞在整个领域与一些有感染，有些已经吞噬数额不等隐球菌细胞。作为电影的进行，细胞ARË所有能动和可以看出移动有关字段。我们能够遵循这些巨噬细胞，看看他们接受什么样的细胞的过程。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52084/52084fig2highres.jpg" width="500" /> 图2:初级小鼠巨噬细胞发生非溶胞胞吐作用的巨噬细胞感染（图A，用白色箭头表示）被认为是处于非溶胞胞吐的开始阶段。吞噬体开始放大（面板BC）和真菌负荷开始转移。然后几个道道的细胞被释放到细胞外空间，可以在面板DE可以看出。第二个被感染的巨噬细胞在F组，由白色箭头指示的，也经历了一个非溶胞胞吐事件（面板生长激素）。在该过程结束时，两巨噬细胞被留为空（小组I）中所示的白色箭头。尺度条为25μm。 <a href="https://www.jove.com/files/ftp_upload/52084/Non-Lytic_Exocytosis.avi" target="_blank">电影2</a> :可视化原代鼠巨噬细胞发生非裂解性胞吐作用在该电影中，很显然，在隐球菌细胞是一种被感染的巨噬细胞的范围内。巨噬细胞变得激动和酵母细胞中可以看出吞噬体中崭露头角。隐球菌细胞，然后快速地擦去到周围环境中。第二个被感染的巨噬细胞发生非裂解性胞吐作用和更酵母细胞被释放到细胞外空间。宿主细胞保持隐球菌胞吐后存活了他们继续往前的能力表示。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52084/52084fig3highres.jpg" width="500" /> 图3:原代鼠巨噬细胞发生细胞-细胞转移的另一个进程内可以使用该协议被捕获ESS是当隐球菌细胞是从1巨噬细胞转移到邻近的巨噬细胞产生的细胞 - 细胞传递。两个受感染的巨噬细胞是在接近彼此以白色箭头（图A）所示，并移动至更接近引发的细胞 - 细胞传递（图B）。由红色箭头指示的细胞间桥被两个巨噬细胞，使得隐球菌细胞的转移（图C，E）中不暴露于周围环境之间形成。转移完成后，将细胞脱离（图D，F）。尺度条为25μm。 <a href="https://www.jove.com/files/ftp_upload/52084/Cell-Cell_Transfer.mov" target="_blank">电影3</a> :可视化感染的巨噬细胞之间的细胞转移有时，巨噬细胞将有利于隐球菌细胞的宿主细胞之间传递的方式进行交互。酵母细胞不暴露向细胞外环境和宿主细胞保持存活。在这部影片中，两名受感染的巨噬细胞产生在两个不同场合穿梭隐球菌细胞从一个巨噬细胞向另一个细胞间的桥梁。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52084/52084fig4highres.jpg" width="500" /> 图4:示意图，显示准备电池。

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

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

我们将介绍如何可视化巨噬细胞C。新型隐球菌 （Cn）的相互作用进行实时，特别强调采用数字光学显微镜非溶胞胞吐过程。单独使用这种技术被感染的巨噬细胞可以研究，以确定这种现象的各个方面。

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

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

visualizing-non-lytic-exocytosis-cryptococcus-neoformans-from

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

11.7K 次观看.  Albert Einstein College of Medicine. 我们将介绍如何可视化巨噬细胞C。新型隐球菌 （Cn）的相互作用进行实时，特别强调采用数字光学显微镜非溶胞胞吐过程。单独使用这种技术被感染的巨噬细胞可以研究，以确定这种现象的各个方面。 

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

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 ...

科研

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 ...

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 ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

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 ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

通过生命内显微镜在斑马鱼胚胎的分孔细菌感染过程中巨噬细胞细胞死亡的可视化

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

可视化吞噬的早期阶段

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

生物学

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

可视化的非溶胞胞吐作用

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