Name: real-time quantification of reactive oxygen species in neutrophils infected with meningitic escherichia coli
Uploaded: 2021-04-20T13:30:00
Description: Watch this Scientific Journal Video about Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli at JoVE.com

这项工作得到了中国国家自然科学基金委员会（31670845、31870832、32000811）和辽宁省杰出教授项目（LJH2018-35）的支持。

本研究中应用的志愿者外周血液经中国医科大学第一医院机构评审委员会批准（#2020-2020-237-2）。
1. 试剂和文化介质的制备
<ol><li>通过添加 8.29 克 NH4Cl、1 克 KHCO 3、37.2 毫克 Na2EDTA 添加到 1 升双蒸馏水中，将 pH 值调整为 7.2-7.4，从而准备红细胞裂解缓冲器。使用 0.22 μm 过滤器过滤细菌。</li>
	<li>通过在RPMI 1640介质中加入5%的胎儿牛血清，并在4°C储存，...

<ol>
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嗜中性粒细胞是人类血液循环中白血球最丰富的成分。它们是先天人类免疫系统中的重要效应细胞，是抵御病原体入侵的第一道防线。ROS的产生代表了11号噬菌体病后嗜中性粒细胞的主要杀菌机制之一。最近的研究表明，一种名为中性粒细胞外陷阱（NET）的中性粒细胞释放出的网状结构也参与了细菌杀灭过程6、11、12。</su...

新生儿细菌性脑膜炎是一种常见的儿科传染病。大肠杆菌（大肠杆菌）与K1胶囊是最常见的革兰阴性病原体引起新生儿细菌性脑膜炎，约占总发病率的80%1，2，3。尽管抗菌化疗和支持性护理取得了进展，但细菌性脑膜炎仍然是发病率高、死亡率最高的疾病之一。
新生儿细菌性脑膜炎的发生通常从致病菌从新生儿的局部病变进入周围循环引起的细菌血症开始，然后通过血脑屏障（BBB）渗透到大脑中，导致脑膜炎。细菌血症的发病取决于细菌与宿主免疫细胞（包括嗜中性粒细胞和巨噬细胞等）之间的相互作用。中性粒细胞占白细胞的50-70%，是抵御细菌感染的第一道防线。在细菌入侵期间，活性嗜中性粒细胞被招募到传染性部位并释放活性氧物种（ROS），包括超氧化物离子、过氧化氢、羟基基和单氧7。ROS对细菌的细胞膜、核酸分子和蛋白质进行氧化反应，导致入侵细菌8的损伤和死亡。线粒体是俄罗斯在真核细胞中生产的主要场所，各种氧化物（如烟酰胺腺苷二氯酰胺磷酸盐（NADPH）氧化酶复合物、脂氧酶系统、蛋白激酶C和环氧酶酶系统）调解了ROS9、10的生产。代表中性粒细胞中主要抗菌机制的ROS生产的实时测量，是研究细菌与宿主相互作用期间宿主防御的有用方法。
在此协议中，受脑膜性大肠杆菌 感染的嗜中性粒细胞中依赖时间的 ROS 生产用荧光 ROS 探头 DHE 进行量化，该探测器由实时荧光微板读卡器检测到。在病原体与宿主相互作用期间，此方法还可用于评估其他哺乳动物细胞中的 ROS 生产。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL polypropylene conical centrifuge tubes</td>
			<td>KIRGEN</td>
			<td>KG2611</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>3025</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>DINGGUO</td>
			<td>DH010-1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Autuomated cell counter</td>
			<td>Bio-rad</td>
			<td>508BR03397</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Carbinet</td>
			<td>Shanghai Lishen</td>
			<td>Hfsafe-1200Lcb2</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-045-701</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Changsha Xiangyi</td>
			<td>TDZ5-WS</td>
			<td></td>
		</tr>
		<tr>
			<td>Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium (DHE)</td>
			<td>MedChemExpress</td>
			<td>104821-25-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Cellmax</td>
			<td>SA211.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heraeus</td>
			<td>Hera Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Beyoitme</td>
			<td>S1819-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS separation Unit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Solarbio</td>
			<td>13292-46-1</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Sangon Biotech</td>
			<td>E600027-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic shaker</td>
			<td>Shanghai Zhicheng</td>
			<td>ZWY-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time quantification of reactive oxygen species in neutrophils infected with meningitic escherichia coli

大肠杆菌 （大肠杆菌）是导致新生儿脑膜炎的最常见革兰阴性细菌。细菌血症的发生和细菌通过血脑屏障的渗透是 大肠杆菌 脑膜炎发展的不可或缺的步骤。活性氧物种（ROS）是中性粒细胞破坏入侵病原体的主要杀菌机制。在本协议中，使用实时荧光微板读卡器检测到的荧光 ROS 探头对感染脑膜 大肠杆菌 的嗜中性粒细胞内 ROS 生产的时间依赖性 ROS 进行量化。该方法还可用于在病原体-宿主相互作用期间评估哺乳动物细胞中的ROS生产。

使用本文概述的协议，嗜中性粒细胞从人类外周血液中分离出来，并装载荧光探头 DHE，以检测针对 E44 感染的 ROS 水平的变化。在这里，我们提供具有代表性的数据，演示由微板读卡器实时确定的 E44 菌株引起的 ROS 生产。通过在 100 的 MOI 中添加 E44 菌株，ROS 水平立即增加，并以时间依赖的方式呈现持续上升趋势（图 1）。通过添加PMA，一个众所周知的中性粒体细胞内ROS的ROS?...

Watch this Scientific Journal Video about Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli at JoVE.com

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

大肠杆菌 是新生儿革兰阴性细菌性脑膜炎的主要原因。在细菌感染期间，中性粒细胞产生的活性氧物种起着主要的杀菌作用。在这里，我们介绍了一种检测中性粒细胞中反应性氧物种以应对 大肠杆菌脑膜炎的方法。

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

大肠杆菌是导致新生儿脑膜炎的最常见克阴性细菌。新生儿细菌性脑膜炎的发生纯粹从细菌血症开始，然后血液中的细菌穿透血脑屏障导致脑膜炎。嗜中性粒细胞是抵御细菌感染的第一道防线。在细菌入侵期间，活性嗜中性粒细胞释放ROS。ROS代表宿主细胞的主要杀菌机制，以摧毁入侵的病原体。测量中性粒细胞中ROS的产生是细菌-宿主相互作用期间启动宿主防御的有用方法。用无菌移液器从盘子中取出一个细菌菌落，放入五毫升脑-心脏输液介质中，在凹凸瓶中含有里芬霉素。在 37 摄氏度下孵育细菌培养，在孵化摇床中 90 RPM 17 小时。在 2000 RPM 下将外周血液样本离心五分钟。离心机后，血液样本分为三层。从下到上，红血球层，白血球层，血浆层。用无菌移液器将白血球层吸到新管中。添加三倍红血球裂解缓冲器。将混合物彻底混合，在室温下放置五分钟。在 2000 RPM 下将管子离心五分钟。完全吸气超自然人并丢弃。重复红血球的裂解过程一到两次，直到沉积物变白。通过用两毫升 PBS 重新悬浮沉积物来清洗细胞。然后在 1000 RPM 下离心管五分钟。用15微升预编码磁细胞分拣缓冲器为1500万个细胞补充沉积物。加入 15 微滑石磁性微珠，彻底混合。在四摄氏度孵化混合物30分钟。在正常情况下，成年人类每微升血液中约有4到1万个白血球。因此，白血球总成本在5毫升的人类外周血液中，接近5000万。15微升磁分拣缓冲器中的50微升磁珠就足够了。中性粒细胞的正常百分比为白血球总数的50%至70%。因此，大约1000万嗜中性粒细胞可以从5毫升的血液中获得。通过在管中加入两个千升磁分拣缓冲器（每 1000 万个细胞）和 4 摄氏度（1200 RPM）的离心机（1200 RPM）10 分钟来清洗细胞。组装磁柱并分离架子。完全丢弃超自然物，用500微升磁分拣缓冲器为多达1亿个细胞补充沉积物。用三毫升磁分拣缓冲器冲洗柱子。将细胞悬架涂在柱子上，使磁珠标记的嗜中性粒细胞附着在柱子上。每次柱储液池为空后，通过添加三毫升缓冲器三次冲洗掉非特定细胞。从磁分离器中取出柱子，并将其放入新管中。将五毫升磁分拣缓冲区添加到列中。使用柱塞推出磁性标记单元。在 1，200 RPM 下将管子离心五分钟。完全吸附超自然物，用一毫升培养介质补充沉积物。使用计数器确定单元格编号。将含有 DHE 荧光探头的培养介质中的细胞浓度调整为每毫升 200 万。将嗜中性粒细胞放在孵化器中 30 分钟以加载 DHE 探头。将细胞悬架分配给一个96井的黑色微板，每口井200微升。打开微板读卡器。将温度设置为37摄氏度，选择荧光读取模式、动能读取类型，设置荧光波长。选择板格式。确定阅读区域。测量荧光强度每五分钟一小时。每次阅读前摇动盘子三秒钟。从孵化器中取出微板。添加 PMA 或 E44 菌株，重复三次。将板放入微板读卡器中，按下启动按钮立即开始检测。使用本文中的协议大纲，中性粒细胞从人类外周血液中分离，并装载荧光探针DHE。通过添加PMA，中性粒细胞内的ROS显著增加，从20分钟增加到40分钟，并在60分钟时达到峰值。但我认为E44菌株，ROS生产立即发生，并增加时间依赖。E44 菌株引起的 ROS 的表达与 PMA 引起的表达相似。在嗜中性粒细胞中检测ROS生产可能有助于进一步研究嗜中性粒细胞的杀菌机制。在此协议中，我们提供了一种更简单的方法，以实时方式检测中性粒细胞中的 ROS 生产。在宿主-细菌相互作用期间，也可用于监控其他宿主细胞类型的 ROS 生产。

Research

JoVE Journal

Immunology and Infection

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

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 ...

科研

免疫与感染

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

实时定量聚合酶链反应检测感染细胞培养中的登革热病毒 rna

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

中性粒细胞中活性氧物种的实时定量感染脑膜性大肠杆菌