Name: isolation of salmonella typhimurium-containing phagosomes from macrophages
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Description: Watch this Scientific Journal Video about Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages at JoVE.com

在罗宾逊实验室的研究得到了来自科隆卓越集团的资助, 这是关于衰老相关疾病的细胞应激反应的支持, 德国科隆大学 (CECAD; 由 DFG 在德国联邦的卓越主动性之内资助和州政府) 和赠款从德意志 Forschungsgemeinschaft (SFB 670), 科隆财富和玛丽亚-Pesch 基金会的德国科隆大学。

所有涉及使用致病性的步骤 . 沙门氏菌 必须在 BSL-2 或更高的生物安全级别设施中进行。 S 的区域性和涂层 鼠伤寒, 以及骨髓源性巨噬细胞 (BMDMs) 的感染必须在层流罩下进行, 以防止污染。 S 的隔离含有吞噬的 鼠伤寒杆菌 可在任何 BSL-2 实验室的工作台上执行. 
 从小鼠体内提取骨髓, 将其分化成巨噬细胞, 是按照动物福利机构指南进行的, ?...

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用于隔离S的新方法本文介绍了用生物素和亲和-共轭磁珠吞噬细菌的方法. 在细胞膜的轻微破坏后, 含有细菌的吞噬可以很容易地用磁性齿条来提取。我们表明, 细菌的标记保留了病原体诱导炎症的能力, 不会改变宿主细胞的吞噬特性。重要的是, 这种方法获得的吞噬丰富的细菌和 endosome/溶标记和缺乏胞浆污染。
与传统的采用蔗糖梯度分离法的体分离方法相比, 该?...

巨噬细胞是循环的专门的吞噬干细胞, 可以检测、吞噬和降解外周组织中的任何外来微粒, 从凋亡细胞到入侵微生物, 如细菌。表面受体介导的病原体特异标记物的识别通常存在于微生物的表面 (称为病原体相关的分子模式或 PAMPs), 巨噬细胞启动一个复杂的细胞膜重组为了包围和吞噬病原体1。
吞噬的病原体然后被巨噬细胞所包含的细胞内囊泡称为体。通过一系列的融合和裂变事件与其他囊泡, 如体和溶酶体, 包含体获得了一套蛋白质, 需要消除 phagosomal 的内容。因此, 体的酶组成在这个过程中是高度可变的, 被称为体成熟的2。
吞噬后不久, multimeric 复合泡 atp 酶 (v atp 酶) 被纳入体膜融合与体3。这个复合体利用 ATP 泵浦质子从胞到腔的体4。体的酸化是必要的为融合事件与其他泡5和为激活许多 pH 依赖性降解酵素6。在体膜上快速组装的另一种 multimeric 酶复合物是氧化酶 (NOX) 复合物。为了产生活性氧 (ROS) 分泌到体腔内, 并大大有助于杀死被吞噬的微生物7, 氮氧化物复合物氧化。
在成熟的初始步骤, 吞噬目前的标记通常如 Rab5 和 Rab7 的早期和晚期体分别与 v 型 atp 酶8的 v-0亚基。吞噬与溶酶体和晚期体的融合导致吞噬病原菌暴露于多种水解酵素, 如蛋白酶蛋白酶、脂肪酶和β-乳糖9。对这些酶的活化也需要管腔的酸化。例如, 蛋白酶 D 的裂解产生活性的短形式是 pH 依赖性的10。这些酶降解病原体, 并调解由巨噬细胞主要组织相容性复合物 (MHC) II 类分子向 T 细胞提出的病原体衍生短肽的产生, 以触发自适应免疫应答11。
因此, 体成熟对先天免疫反应至关重要, 并与免疫系统的先天和适应性武器联系在一起。这并不奇怪, 病原体已经进化的战略, 以克服由巨噬细胞通过上述的体成熟的过程。例如, 胞内细菌结核分枝杆菌和军团肺通过抑制 v atp 酶的组装和相应的流明酸化, 防止体成熟,12,13.其他细菌如李斯特菌或志贺氏杆菌福氏诱导体膜中的孔隙形成, 以逃逸到胞14,15。另一方面,沙门氏菌 enterica螺旋体鼠伤寒杆菌 (S。沙门氏菌)能够修改液泡内体的属性, 将其转换为其复制16的合适位置。此功能使S.沙门氏菌是研究病原体介导的体成熟干预的一个非常有趣的模型。
S. 鼠伤寒是一种在人类中引起胃肠炎的兼胞内细菌。在侵入固有后, S.鼠伤寒杆菌快速检测和吞噬的巨噬细胞, 并包含在吞噬17。一些报告以前曾描述过S.沙门氏菌-包含吞噬目前的制造商为体和溶酶体18, 和其他研究发现体-溶融合阻止后, S.鼠伤寒病毒感染19。
最初, 体成熟于S.用免疫荧光显微镜对鼠伤寒杆菌感染进行了研究。在分离细菌的吞噬技术的发展使更准确的研究体的内容, 从 endosome 和溶标记。迄今为止, 用于分离细菌的吞噬的主要方法是蔗糖梯度的亚细胞分馏18,20。然而, 这种方法需要多个离心步骤, 可以造成机械损伤的吞噬, 可能会影响稳定的 phagosomal 组分 (蛋白质和血脂), 是费时。此外, 它还需要使用 ultracentrifuge: 一个专门的设备, 这是无法进入每个实验室。
最近, 一种新的方法已被用于分离细菌含有吞噬, 其中细菌病原体被标记为化 lipopetide (Lipobiotin), 后来提取使用亲和共轭磁珠21.我们提出了一种替代的补充方法, 标记细菌表面胺类大分子与 NHS-生物素后, 亲和共轭磁珠。该方法获得的吞噬在 endosome 和溶标记物中具有很高的丰富性, 可用于多种测定, 从蛋白质分析到学分析。此外, 它不需要专门的设备, 如超速。此外, 通过消除离心步骤, 吞噬的机械损伤和使用的时间都大大减少。这种方法可以很容易地适应隔离的吞噬含有其他细菌, 如革兰氏阳性的金黄色葡萄球菌, 也包括在这份手稿。总之, 此方法用于隔离S.含有吞噬的鼠伤寒杆菌是一种简单、cost-effective 且耗时较传统的蔗糖梯度分离法, 离心, 呈现高浓度的含菌吞噬。

<table>
	<tbody>
		<tr>
			<td>EZ-Link NHS Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>20217</td>
			<td></td>
		</tr>
		<tr>
			<td>FluidMag Streptavidin</td>
			<td>Chemicell</td>
			<td>4205</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Carl Roth</td>
			<td>9156.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>A537.4</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Carl Roth</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Carl Roth</td>
			<td>4175.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43816</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase inhibitor cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>1861280</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin B</td>
			<td>Sigma</td>
			<td>C6762</td>
			<td></td>
		</tr>
		<tr>
			<td>DYNAL or DynaMag Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartSpec 3000 Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial loop (10&#181;l)</td>
			<td>Sarstedt</td>
			<td>86.1562.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella enterica serovar Typhimurium SL1344</td>
			<td>Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Biochrom</td>
			<td>FG1415</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom</td>
			<td>L1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5-streptavidin</td>
			<td>Invitrogen</td>
			<td>SA1011</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-beta-actin antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-47778</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mCherry antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-34974</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rab5 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-46692</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rab7 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-10764</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-v-ATPase (V0) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-28801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-v-ATPase (V1) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20943</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cathepsin D antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-6486</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Tomm20 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-17764</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-calnexin antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-46669</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20357</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of salmonella typhimurium-containing phagosomes from macrophages

鼠伤寒沙门氏菌是一种在人类中引起胃肠炎的兼胞内细菌。入侵后的叶片固有, S.鼠伤寒杆菌细菌被巨噬细胞快速检测和吞噬, 并被称为吞噬的囊泡, 以使其降解。隔离S.包含吞噬的沙门氏菌已被广泛用于研究S.沙门氏菌感染会改变体成熟的过程, 以防止细菌降解。采用蔗糖梯度离心法对含菌吞噬进行了分离。然而, 这个过程是费时的, 需要专门的设备和一定程度的灵巧。这里描述的是一个用于隔离 S 的简单而快速的方法.鼠伤寒-含有吞噬从巨噬细胞通过涂层细菌与生物素-亲和共轭磁珠。通过这种方法获得的吞噬可以悬浮在任何选择的缓冲液中, 使分离的吞噬的使用范围广泛, 如蛋白质、代谢物和脂质分析。总之, 此方法用于隔离S.含有吞噬的鼠伤寒杆菌是特定的、高效的、快速的, 需要最少的设备, 并且比传统的蔗糖梯度离心方法更具通用性。

通过本议定书隔离含有细菌的吞噬, 需要将细菌的法作为第一步。因此, 我们评估了S.的有效性鼠伤寒杆菌法通过共聚焦显微镜分析 BMDMs 感染化 mCherry-S。沙门氏菌标记为 Cy5-Streptavidin。简言之, BMDMs 在本协议中与 mCherry-S一起被感染。沙门氏菌先前化, 但不与亲和共轭磁珠一起孵育。在37° c 的30分钟孵育后, 细胞用温热的 pbs 洗涤, 用4% ?...

Watch this Scientific Journal Video about Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages at JoVE.com

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

我们在这里描述了一个简单和快速的方法, 以隔离的鼠伤寒沙门氏菌-含有吞噬从巨噬细胞涂层细菌与生物素和亲和。

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. ...

Research

JoVE Journal

Immunology and Infection

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

科研

免疫与感染

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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 ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

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

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. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

慢性沙门氏菌感染诱发肠纤维化

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...