Name: investigating the phagocytosis of leishmania using confocal microscopy
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Description: Watch this Scientific Journal Video about Investigating the Phagocytosis of Leishmania using Confocal Microscopy at JoVE.com

我们感谢巴西菲奥克鲁斯巴伊亚的贡萨洛·莫尼兹研究所和显微镜系的援助。这项工作得到了国际志愿者协会-FIOCRUZ号79700287000的支持，P.S.T.V.持有CNPq（305235/2019-2）的研究生产力补助金。质粒由加利福尼亚州多伦多大学的毛里西奥·特雷比兹尼克提供。作者要感谢安德里斯·沃尔特的英语语言修订和手稿文案编辑帮助。

细胞是在国家研究伦理委员会批准程序后从健康供体获得的（ID：94648218.8.0000.0040）。
1. 细胞培养
<ol><li>人单核细胞来源的巨噬细胞 注意：要获得用于 体外 分化为巨噬细胞的人单核细胞，请从健康供体收集血液并纯化D.英语和B.R.Andersen21描述的外周血单核细胞（PBMC）。<ol><li>收集外周血（50毫升）后，将其倒入肝素化管中，然后在室温下在磷酸盐?...

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利什曼原虫 - 巨噬细胞相互作用是一个复杂的过程，涉及几个可能影响疾病发展的步骤5。为了更好地了解未被置 信化的利什曼原虫 和宿主细胞相互作用所涉及的机制，我们描述了一种方案，该方案采用共聚焦荧光显微镜来评估 利什曼原虫 感染早期至晚期的吞噬作用。使用涉及两个或多个荧光团的荧光技术来研究细胞生物学机制，包括免疫标记和荧光标记?...

利什曼病是一种被忽视的热带病，由 利什曼原虫属的原生动物寄生虫引起，导致脊椎动物宿主的广泛临床表现，包括皮肤利什曼病、皮肤黏膜利什曼病和内脏利什曼病1。世界卫生组织（WHO）估计，超过10亿人处于危险之中，每年报告的新病例超过100万2。
利什曼原虫 属是专性细胞内原生动物，在宿主细胞内存活，包括单核细胞、巨噬细胞和树突状细胞3. 利什曼原虫 - 巨噬细胞相互作用是一个复杂的过程，通过直接相互作用或通过涉及补体受体的调理作用涉及多个宿主细胞受体和寄生虫配体4，5。经典的表面受体，如CR1，CR3，甘露糖 - 岩藻糖，纤连蛋白，Toll样和清道夫受体，介导寄生虫附着到巨噬细胞6，7，8。这些受体识别 利什曼原虫表面的分子，包括63 kDa糖蛋白（gp63）和糖脂脂脂磷酸聚糖（LPG）9。这些是前鞭毛虫表面最丰富的分子，在破坏宿主免疫反应中起着至关重要的作用，有利于在哺乳动物细胞中建立寄生虫感染10。在寄生虫表面配体与巨噬细胞受体结合后，F-肌动蛋白积聚在哺乳动物细胞表面，在寄生虫被吞噬时围绕它们。随后，这导致形成寄生虫诱导的隔室，称为寄生液泡（PV），其呈现吞噬溶酶体特征11。一旦进入这些吞噬溶酶体，寄生虫就会经历生存和繁殖所必需的几种改变3。
PV的生物发生是一种高度调控的膜运输过程，对这种病原体12的细胞内存活至关重要。该区室的形成是由吞噬体和宿主内吞途径的区室之间的连续融合事件引起的。经典细胞生物学研究表明，PV的成熟涉及单体G蛋白Rab 5和Rab 7蛋白的获取，这主要与早期和晚期内体成熟有关，分别是13。此外，这些区室获取溶酶体相关膜蛋白1和2（LAMP 1，LAMP 2），溶酶体膜的主要蛋白质成分和微管相关蛋白1A / 1B-轻链3（LC3），自噬体标记物14。尽管有明显的相似性，但PV形成15，16 的动力学和这些区室的形态因 利什曼原虫 物种而异。例如，由 墨西哥乳杆菌 或 亚马逊乳杆菌 引起的感染诱导形成含有大量寄生虫的大隔室17。相比之下，其他物种，如 巴西乳杆菌 和 婴儿乳杆菌， 形成较小的液泡，通常每个液泡18中只含有一到两个寄生虫。
尽管对宿主细胞-利什曼原虫 相互作用有这种了解，但由宿主受体和寄生虫配体之间接触引发的初始事件尚未完全阐明。已知这些事件是寄生虫感染结果的决定因素，并且取决于寄生虫种类，招募以识别寄生虫的宿主细胞受体的类型以及巨噬细胞信号通路的激活19，20。因此，必须鉴定参与 利什曼原虫诱导的PV的生物发生的分子，并确定这些分子在感染建立和结果中的作用。在这里，我们描述了一种监测 利什曼原虫吞噬过程中发生的早期事件的方法，包括结合，内化，吞噬体形成和成熟。这项工作有助于澄清PLC，Akt，Rab5，Rab7和LC3参与由不同 利什曼原虫 物种诱导的PP的形成。重要的是，该方案可用于研究参与PV成熟的其他蛋白质的参与。未来的研究将扩大利 什曼原虫 - 宿主细胞相互作用所涉及的机制的知识，并有助于设计新的化疗策略。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488-conjugated goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>Tem varios no site</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-LC3 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB600-1384</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>CellStripper</td>
			<td>Corning</td>
			<td>25-056-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red (CMTPX) Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>Centr&#237;fuga</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Isofarma</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal fluorescence microscope (Leica SP8)</td>
			<td>Leica</td>
			<td>Leica SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope (Olympus Lx73)</td>
			<td>Olympus</td>
			<td>Olympus Lx73</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750045</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (N- 2-hydroxyethyl piperazine-N&#8217;-2-ethane-sulfonic acid)</td>
			<td>Gibco</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma</td>
			<td>NS02L</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector solution</td>
			<td>Lonza</td>
			<td>VPA-1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol myristate acetate (PMA)</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer solution (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade kit</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Thermo Fisher Scientific</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect medium</td>
			<td>Sigma</td>
			<td>S0146</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating the phagocytosis of leishmania using confocal microscopy

吞噬作用是一个精心策划的过程，涉及不同的步骤：识别，结合和内化。专业吞噬细胞通过吞噬作用吸收 利什曼原虫 寄生虫，包括通过多个宿主细胞受体识别寄生虫表面上的配体。 利什曼原虫 与巨噬细胞膜的结合通过补体受体1型（CR1）和补体受体3型（CR3）和模式识别受体发生。脂磷聚糖（LPG）和63 kDa糖蛋白（gp63）是参与巨噬细胞 - 利什曼原虫 相互作用的主要配体。在宿主细胞受体最初识别寄生虫配体之后，寄生虫在寄生液泡内化，存活并繁殖。 利什曼原虫诱导的液泡的成熟过程涉及从细胞内囊泡中获取分子，包括单体G蛋白Rab 5和Rab 7，溶酶体相关膜蛋白1（LAMP-1），溶酶体相关膜蛋白2（LAMP-2）和微管相关蛋白1A / 1B-轻链3（LC3）。
在这里，我们描述了使用共聚焦显微镜评估 利什曼原虫 与宿主细胞相互作用期间发生的早期事件的方法，包括（i）结合（ii）内化和（iii）吞噬体成熟。通过增加围绕这些感染结果决定因素的知识体系，我们希望提高对 利什曼原虫 感染发病机制的理解，并支持最终寻找新的化疗靶点。

本报告旨在评估从呈现 巴西乳杆菌 -LCL或 巴西乳杆菌-DL形式的CL的患者中分离出的巴西 乳杆菌吞噬过程中发生的早期事件。我们首先评估了 人单核细胞来源的巨噬细胞对 巴西猪笼草-LCL或巴西猪笼草-DL的结合和吞噬作用。数据显示， 巴西乳杆菌-LCL和 巴西乳杆菌-DL与巨噬细胞的结合相似（图1）。此外，没有观察到宿主细胞对 <...

Watch this Scientific Journal Video about Investigating the Phagocytosis of Leishmania using Confocal Microscopy at JoVE.com

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

利 什曼原虫 感染中与吞噬作用相关的机制仍然知之甚少。在这里，我们描述了评估 利什曼原虫 与宿主细胞相互作用期间发生的早期事件的方法。

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

利什曼原虫感染中吞噬作用的相关机制仍然知之甚少。在这里，我们描述了评估利什曼原虫与宿主细胞相互作用期间早期固化状态的方法。该技术作为主要优点，允许研究利什曼原虫吞噬作用的不同状态和几种蛋白质的参与。值得注意的是，这种技术也可以扩展到其他类型的研究，包括细菌、酵母菌感染，或者被其他类型的细胞吞噬。尝试这种技术的人应该注意细胞暴露于核苷酸溶液的时间。此外，我们建议开始测试转染，最多使用两个质粒。演示该程序的将是来自我们实验室的博士生Amanda Reboucas。首先，在带有13毫米玻璃盖玻片的24孔板上接种每孔500微升RPMI的020万个THP-1细胞或人单核细胞衍生巨噬细胞。在37摄氏度下在5%二氧化碳下孵育24小时，用0.9%盐水洗涤细胞两次，并将细胞在完整的RPMI培养基中孵育。向细胞中加入固定相利什曼原虫种属，以10个寄生虫与一个宿主细胞的比例，然后，在4摄氏度下以720倍g离心10分钟。将细胞在四摄氏度下孵育五分钟后，用0.9%盐水溶液洗涤细胞两次，以去除任何未内化的前鞭毛。将细胞在补充的RPMI培养基中在37摄氏度下孵育一小时，然后用4%PFA固定细胞15分钟。接下来，使用首选的封片介质安装盖玻片。然后，在荧光显微镜下在随机场中计数至少400个细胞。为了研究利什曼原虫诱导的PV生物发生，通过将1500万个THP-1细胞接种在75平方厘米的组织培养瓶中，将1500万个THP-1细胞接种在含有10毫升完全RPMI培养基（补充有100纳克/毫升PMA）和50微摩尔至巯基乙醇中48小时，开始用质粒转染THP-1细胞48小时。然后，在0.9%盐水溶液中洗涤细胞一次，并使用非酶细胞解离溶液分离细胞。然后，在室温下以250倍g离心5分钟。接下来，将THP-1细胞重悬于一毫升RPMI培养基中，并在Neubauer室中进行计数。在室温下再次以250倍g离心细胞10分钟，弃去上清液。将200万个细胞重悬于100微升的核苷酸溶液中，并与0.5微克质粒包被孵育，以获得用荧光蛋白标记的目标蛋白。将含有THP-1细胞和核酸的悬浮液转移到核素比色皿中。然后，使用核苷程序Y一转染细胞。在 500 微升 RPMI 培养基中回收转染的细胞和种子，该培养基在 24 孔板上使用13毫米玻璃盖玻片。将细胞和完全RPMI培养基在37摄氏度下孵育0.5、2、4、6、12和24小时。将 13 毫米玻璃盖玻片置于室温下，并在采集前至少 30 分钟保护它们免受光照。用吸收性纸巾清洁盖玻片。接下来，向物镜中加入一滴浸油，然后添加幻灯片。向上移动物镜，直到油接触载玻片。观察并调整显微镜上的焦点，然后选择带油的63x物镜选项。打开 LIQA 程序并调整 488、552 和 405 波长的激光器。选择图像分辨率为 1024 x 1024。单击实时按钮后，设置 Z 堆栈并按开始选项。等待图像采集，然后在工具中选择选项“最大投影”。保存实验，并将 TIFF 格式的图像导出到计算机。结果表明，巴西利什曼原虫LCL和巴西利什曼原虫DL与巨噬细胞相似。此外，在宿主细胞吞噬巴西利什曼原虫LCL和巴西利什曼原虫DL吞噬作用方面没有观察到差异。在感染细胞中比较了LC3募集到由巴西利什曼原虫LCL或巴西利什曼原虫DL诱导的寄生液泡或PV。感染4小时后，观察到巴西利什曼原虫LCL和巴西利什曼原虫DL感染巨噬细胞中LC3修饰PV的百分比相似。因此，结果表明，巴西利什曼原虫LCL和巴西利什曼原虫DL在LC3募集PV的结合，吞噬作用和生物发生过程中与巨噬细胞发生类似相互作用。代表性的显微图像显示了用PLC-GFP，Rab5-GFP，Rab7-GFP质粒有效转染THP-1细胞。尝试此协议的人必须注意温度孵育并保护显微镜样品免受所有类型的光线照射。调节诱发利什曼原虫吞噬作用的已鉴定蛋白质的表达将证明这些蛋白质在利什曼原虫感染结果中的重要性。我们可以将这种技术扩展到不同类型的病理学研究和与寄生虫建立和靶标识别相关的机制，以制定治疗策略。

Research

JoVE Journal

Immunology and Infection

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

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

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 ...

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

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 ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Live-cell Video Microscopy of Fungal Pathogen 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 ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages 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 ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染：肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

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 ...

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

使用共聚焦显微镜研究利什曼原虫的吞噬作用