Name: visualization of macrophage lytic cell death during mycobacterial infection in zebrafish embryos via intravital microscopy
Uploaded: 2019-01-09T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy at JoVE.com

我们感谢温子龙博士分享斑马鱼菌株，斯特凡·奥勒斯博士和大卫·托宾博士分享马兰姆相关资源，何跃鹏协助进行图体准备。这项工作得到了国家自然科学基金（81801977）（B.Y.）、上海市卫生委员会杰出青年培训计划（2018YQ54）（B.Y.）、上海航海项目（18YF1420400）和上海结核病重点实验室开放基金（2018KF02）（B.Y.）的支持。

斑马鱼是在标准条件下养殖的，符合实验室动物动物福利伦理审查准则（GB/T 35823-2018）。本研究的所有斑马鱼实验均获批（2019-A016-01），在复旦大学上海公共卫生临床中心进行。
1. M. 马里南单细胞接种制剂 （图 1）
<ol><li>从 -80°C 解冻 Cerulean-荧光M. 大麻甘油，用 10% （v/v） OADC、0.25% 甘油和 50 μg/mL 湿霉素接种 7H10 琼脂板。在32°C下孵育板?...

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该协议描述了分管感染期间巨噬菌体死亡的可视化。根据细胞膜完整性等因素，感染驱动细胞死亡可分为凋亡和裂解细胞死亡24、25。Lytic细胞死亡对生物体的压力比凋亡更大，因为它触发强烈的炎症反应24，25。 由于要求高时空分辨率、适当的共聚焦显微镜设置和强烈的转基因表达，很难观察体内的溶血细胞?...

分体菌感染已被证明会导致宿主免疫细胞死亡1。例如，衰减菌株会触发巨噬细胞凋亡并控制感染。然而，毒株会触发溶血细胞死亡，导致细菌传播1，2。考虑到这些不同类型的细胞死亡对宿主抗分体细菌反应的影响，需要详细观察体内分管感染期间的巨噬细胞死亡。
测量细胞死亡的常规方法是使用死细胞污渍，如安宁五，TUNEL，或丙氨酸橙/碘化钠染色3，4，5。然而，这些方法无法揭示体内细胞死亡的动态过程。活成像6已经促进了体外细胞死亡的观察。然而，结果是否准确模拟生理状况仍不清楚。
斑马鱼是研究宿主抗分枝杆菌反应的极好模型。它有一个高度保守的免疫系统，类似于人类，一个容易操纵的基因组，和早期胚胎是透明的，允许活成像7，8，9。感染M.Marinum后，成年斑马鱼形成典型的成熟肉芽肿结构，胚胎斑马鱼形成早期肉芽肿等结构9，10。先天免疫细胞-细菌相互作用的动态过程在斑马鱼M.Marinum感染模型11、12中已经探索。然而，由于高时空分辨率要求，有关先天免疫细胞死亡的细节在很大程度上仍未定义。
在这里，我们描述了如何可视化由体内分体细菌感染引发的巨噬细胞细胞死亡过程。此协议也可以应用于可视化发育和炎症期间体内的细胞行为。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Tween-80</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>Solarbio</td>
			<td>YA0552</td>
			<td></td>
		</tr>
		<tr>
			<td>10% OADC</td>
			<td>BD</td>
			<td>211886</td>
			<td></td>
		</tr>
		<tr>
			<td>3-aminobenzoic acid</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>5 &#956;m filter</td>
			<td>Mille X</td>
			<td>SLSV025LS</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#956;l/ml hygromycin</td>
			<td>Sangon Biotech</td>
			<td>A600230</td>
			<td></td>
		</tr>
		<tr>
			<td>7H10</td>
			<td>BD</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>7H9</td>
			<td>BD</td>
			<td>262310</td>
			<td></td>
		</tr>
		<tr>
			<td>A glass bottom 35 mm dish</td>
			<td>In Vitro Scientific</td>
			<td>D35-10-0-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech</td>
			<td>A60015</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP5 II</td>
			<td></td>
		</tr>
		<tr>
			<td>Enviromental Chamber</td>
			<td>Pecon</td>
			<td>temp control 37-2 digital</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microloader</td>
			<td>Eppendorf</td>
			<td>No.5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>Bioland Scientific LLC</td>
			<td>7105P</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sangon Biotech</td>
			<td>A100854</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Keelrein</td>
			<td>PH-140(A)</td>
			<td></td>
		</tr>
		<tr>
			<td>M.marinum</td>
			<td></td>
			<td></td>
			<td>ATCC BAA-535</td>
		</tr>
		<tr>
			<td>Microinjection needle</td>
			<td>World Precision Instruments</td>
			<td>IB100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Eppendorf</td>
			<td>Femtojet</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>NARISHIGE</td>
			<td>MN-151</td>
			<td></td>
		</tr>
		<tr>
			<td>msp12:cerulean</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 25470057; 27760340</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU</td>
			<td>Sigma</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Single concavity glass microscope slide</td>
			<td>Sail Brand</td>
			<td>7103</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>SCICNTZ</td>
			<td>JY92-IIDN</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer (OD600)</td>
			<td>Eppendorf AG</td>
			<td>22331 Hamburg</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>OLYMPUS</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(mfap4:eGFP)</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 30742890</td>
		</tr>
		<tr>
			<td>Tg(coro1a:eGFP;lyzDsRed2)</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 31278008</td>
		</tr>
		<tr>
			<td>Tg(mpeg1:LRLG;lyz:eGFP)</td>
			<td></td>
			<td></td>
			<td>Ref.: PMID 27424497; 17477879</td>
		</tr>
	</tbody>
</table>

visualization of macrophage lytic cell death during mycobacterial infection in zebrafish embryos via intravital microscopy

斑马鱼是研究先天免疫细胞行为的优秀模型生物体，由于其透明性，在早期发育过程中完全依赖于其先天免疫系统。斑马鱼分枝杆菌（M.Marinum）感染模型在研究宿主对分枝杆菌感染的免疫反应方面已经确立。有人建议，不同的巨噬细胞死亡类型将导致分管感染的不同结果。在这里，我们描述了一个协议，使用生命内显微镜来观察斑马鱼胚胎在M.Marinum感染后巨噬细胞死亡。斑马鱼转基因线，专门标记巨噬细胞和嗜中性粒细胞通过肌肉内显微注射荧光标记M.Marinum在中脑或躯干感染。受感染的斑马鱼胚胎随后安装在低熔胶琼脂上，并通过X-Y-Z-T尺寸的共聚焦显微镜观察。由于长期实时成像需要使用低激光功率来避免光漂白和光毒性，因此强烈建议使用强表达转基因。该协议有助于可视化体内的动态过程，包括免疫细胞迁移、宿主病原体相互作用和细胞死亡。

分枝杆菌感染可以触发不同的宿主响应，根据感染途径。在此协议中，斑马鱼胚胎通过肌肉内微注射荧光标记的细菌进入中脑或躯干（图3），并通过共聚焦活成像进行观察。通过这两种途径的感染将局部限制感染，导致先天免疫细胞招募和随后的细胞死亡。
可视化先天免疫细胞死亡的细节是具有挑战性的。溶血细胞死亡发生在很短的时间窗口，需要高?...

Watch this Scientific Journal Video about Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy at JoVE.com

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

该协议描述了一种技术，用于可视化在胚胎斑马鱼的巨噬行为和死亡在分枝杆菌感染期间。包括制备细菌、胚胎感染和生命内显微镜的步骤。在涉及感染或无菌炎症的类似情况下，此技术可应用于观察细胞行为和死亡。

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 ...

该协议可以清楚地区分分枝杆菌感染期间的巨噬细胞死亡模式。通过并行观察多个胚胎，它大大增加了捕获整个巨噬细胞细胞死亡过程的概率。该协议也适用于在涉及感染或无菌炎症的类似情况下观察细胞死亡和其他细胞行为。在扩展的实时成像过程中，保持激光的强度尽可能低，以避免光漂白和毒性是非常重要的。根据手稿接种后，在3000次G下离心培养10分钟，将分枝杆菌收集为颗粒。丢弃所有，但300微升的上一液，并重新悬浮颗粒。加入3毫升的7H9介质与10%甘油进一步重新悬浮颗粒，然后在水浴中声波悬浮液在100瓦，15秒开和15秒关闭，共两分钟，以实现一个单一细胞均质。将细菌悬浮液转移到10毫升注射器中，并通过5微米过滤器去除任何细菌团块。使用分光光度计测量悬浮液的光学密度，并使用含有 10%甘油至 OD 600 的 7H9 介质稀释。将悬浮液分成10微升等分，储存在负80摄氏度的冰柜中，供进一步使用。首先，在95摄氏度的加热块中加热气，直到其完全熔化。将气糖放入45摄氏度的加热块中，保持液体形式的气糖。要安装在树干区域肌肉感染，创建底部的阿加罗斯层，通过体积 agarose 均匀地将 0.5 毫升 1% 的重量均匀地浇注到玻璃幻灯片上，以创建底部的 agarose 层。将幻灯片放在冰架或冷表面上三分钟，以凝固。麻醉斑马鱼胚胎后，将多达60个斑马鱼胚胎放在底部的阿加罗斯层上，并小心地将它们布置成两排。用纸巾去除底部加糖层上剩余的水，然后按体积加糖添加 0.3 毫升 0.5% 的重量以创建上层。确保胚胎完全嵌入在糖中。将玻璃滑梯再次返回到冰盒中，以凝固加糖。通过额外的 E3 蛋水覆盖表面，保持气糖的顶层湿润。接下来，将微注射器和微操纵器调整到微注射的正确位置和设置。使用微加载器将三根微升的准备好的细菌培养剂转移到准备好的针头中。缓慢而小心地移液，以避免形成气泡。将 100 CFU 注入中继区域。微注射后，用塑料移液器小心地将斑马鱼胚胎冲洗成新鲜的蛋水中。要安装中脑感染，使用塑料移液器将四到六个三分五角的胚胎移植到红糖中。用10个量针小心地将每个胚胎的头部向上放置。一旦所有胚胎位置固定，将玻璃幻灯片转移到冰盒或冷表面，让红糖凝固。将 500 CFU 注入中脑。微注射后，用塑料移液器小心地将斑马鱼胚胎冲洗成新鲜的蛋水中。一旦阿加罗斯完全凝固，用一层蛋水盖住加糖。设置环境室后，将 35 毫米玻璃底盘与斑马鱼放在环境室中。打开 405 二极管，在 20% 的功率下打开 argon，以及 DPSS 561 纳米激光器。在频谱设置中设置适当的激光功率。选择 XYZ 顺序扫描采集模式，将图像格式设置为 512 x 512 像素。切换到实时数据模式，定位第一个斑马鱼的位置，并标记开始和结束 Z 位置。对剩余的每个胚胎重复此过程。在程序结束时添加暂停。定义程序的循环和循环，并保存文件。在这项研究中，我们利用先前报道的转基因共生1a：eGFP;lyzDsRed2，以及转基因mpeg1loxP;DsRed：loxPeGFP;lyz：eGFP，来区分体内的巨噬细胞和嗜中性粒细胞。大量被细菌感染的巨噬细胞变得圆，并表现出运动性降低，最终的细胞质肿胀，细胞膜破裂，细胞质含量迅速传播。紫外线辐射巨噬细胞显示典型的凋亡细胞表型，如细胞收缩、核分裂和染色质凝结。还观察到巨噬细胞积极噬菌体和传播M.merinum。然而，嗜中性粒细胞的吞噬能力有限，并迅速经历了细胞细胞死亡，没有明显的细菌感染。结合强大的基因编辑工具，该协议可为进一步了解多种因素对体内宿主-病原体相互作用的影响提供一个有效的平台。

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Inguinal Lymph Node

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

科研

免疫与感染

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. ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Study of Phagolysosome Biogenesis in Live Macrophages

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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

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

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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 ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

饱和脂肪酸诱导神经酰胺相关巨噬细胞死亡

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...