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...

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 border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sabouraud Dextrose Agar</td>
			<td style="width:183px;">BD</td>
			<td style="width:227px;">DF0109-17-1</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sabouraud Dextrose Broth</td>
			<td>BD</td>
			<td>DF0382-17-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dubelcco&#39;s Modified Eagle&#39;s Medium(DMEM)</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum ( FCS)&#160;</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NCTC 109</td>
			<td>Invitrogen</td>
			<td>21340-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES Buffer</td>
			<td>Corning Cellgro</td>
			<td>25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3003 Tissue Culture Petri Dishes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellStripper</td>
			<td>Corning Cellgro</td>
			<td>25-056-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mat-tek Glass Bottom Culture Dishes</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>Sigma</td>
			<td>L3137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-g</td>
			<td>Roche</td>
			<td>NC 9222016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAb 18B7</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160; Non-commerical antibody produced in our lab</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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...

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 ...

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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

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

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

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

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

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

科研

免疫与感染

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 ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

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

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

荧光寿命成像显微镜观察细胞内圈套的贩运

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