我们感谢马库斯Schnare教授为我们提供了TLR2 - / -和TLR2 / 4 - / -小鼠和卡斯滕Kirschning教授博士TRIF / MyD88的- / -小鼠。这项工作的份资助的德国联邦联邦教育与研究部（E:生物miRSys - FKZ 0316175B，E:地中海CAPSYS - FKZ 01X1304E; http://www.bmbf.de/），德意志研究联合会（SFB / TR-84; http://www.sfb-tr84.de/）和Hessisches Ministerium献给Wissenschaft UND孔斯特（LOEWE医疗RNomics - FKZ 519/03 / 00.001-（0003）; http://www.proloewe.de/medicalrnomics）所有BS。

1.准备中型和琼脂平板<ol><li>准备1升肉汤培养基（YEB）的。溶解10克ACES和在900毫升的蒸馏水10g酵母提取物。将pH调节至6.9，用KOH（5N）。添加L-半胱氨酸（0.4克在10毫升的蒸馏水中）和10毫升的Fe的10毫升（NO 3）3 x9H 2 O（0.25克10毫升蒸馏水）。填充到1L蒸馏水，并过滤灭菌溶液（孔径:0.22微米）。保存在4℃。 </li><li>制备缓冲炭酵母提取物（BCYE）琼脂平板上。溶解10克ACES和在900毫升的蒸馏水10g酵母提取物。将pH调节至6.9，用KOH（5N）。加入琼脂15克和2.5克活性炭。填充到1升用蒸馏水和高压釜中。 <ol><li>添加L-半胱氨酸的10毫升（0.4克在10毫升的蒸馏水中）和10毫升的Fe（NO 3）3 x9H 2 O（0.25克10毫升的蒸馏水中，两个过滤灭菌通过0.22μm的孔）到冷却的BCYE（约50℃）。在4℃下倾板和存储。 </li></ol></li></ol> 2.培养嗜肺军团菌 <ol><li>上BCYE琼脂板上传播嗜肺军团菌菌株科比（野生型，WT）和在37°C孵育他们3天。接种10毫升YEB中的一个外径的0.3与来自预培养板嗜肺军团菌 600;孵育在6小时的旋转摇床（150rpm）下在37℃下的细菌。 </li><li>通过在血琼脂平板扩频100μL的悬浮液的验证液体培养的纯度。在37℃孵育过夜。 </li><li>剩余的液体培养中添加90毫升新鲜的YEB培养基，并孵育在旋转摇动器（37℃和150rpm下）到达的OD 3.0-3.5 600，其需要大约16-20小时。 </li></ol> 3.准备和量化嗜肺军团菌的OMV 注:执行以下所有离心步骤小号在无菌条件下，并在4℃。 <ol><li>离心培养液以4,000×g离心20分钟以沉淀细菌。将上清转移到新的离心管中，弃去细菌沉淀，并重复离心分离（4000×g离心20分钟）。重复此步骤一次。 </li><li>无菌过滤器的剩余的上清液两次（孔径:0.22微米）。传送无菌上清液超速离心管和超速离心机以100,000×g离心3小时。 </li><li>倒出上清液并丢弃它。重悬在无菌磷酸盐缓冲盐水（PBS）和超速离心（100,000×g离心3小时）的监查沉淀以除去污染性蛋白和LPS。 </li><li>弃上清，悬浮颗粒OMV在无菌PBS 500微升。条纹20微升在血琼脂平板并在BCYE琼脂板上以排除所制备的小泡的细菌污染。孵育3天（两个血琼脂平板过夜，并在BCYE琼脂板上在376; C）。 </li><li>量化从使用根据制造商的说明一个双金鸡宁酸测定法OMV制备得到的蛋白质量。 注:L的毫升100 菌培养物的浓度通常为1微克/微升。存放在-20°C的准备和量化的OMV。 </li></ol> 4.预巨噬细胞治疗<ol><li>制备THP-1细胞。 注:THP-1是从白血病患者来源的单核细胞系。 <ol><li>添加2×10 5 THP-1细胞每24个孔，并通过加入20nM的佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）为巨噬细胞样细胞区分。孵育在37℃下24小时。 </li><li>用新鲜培养基的500微升更换培养基并孵育另外24小时对THP-1细胞的最佳培养基组成的RPMI 1640高葡萄糖补充有10％胎牛血清的。 </li></ol></li><li>隔离的鼠骨髓衍生巨噬细胞（mBMDM），如在参见描述ENCE 20。 </li><li>治疗THP-1巨噬细胞或mBMDM用OMV的。 <ol><li>解冻在步骤3中制备的OMV的和根据它们的蛋白量（0.1，1和10微克/毫升）的人或鼠巨噬细胞添加它们。孵育OMV的巨噬细胞，在37℃下进行至少20小时。使用上清液ELISA或第5步前进。 </li></ol></li></ol> 5.感染巨噬细胞和评估细菌复制与菌落形成单位（CFU）分析<ol><li>从步骤2.1使用嗜肺 ，经预处理的THP-1细胞或mBMDM来自步骤4.3，和不进行预处理的巨噬细胞作为对照（2×10 5/24个孔）。不交换的媒介。 </li><li>感染THP-1细胞与嗜肺军团菌科比WT和mBMDM与嗜肺科比的鞭毛蛋白缺乏突变体（两者以感染复数为0.5（MOI）; 1×10 5 嗜肺 / 24孔）并孵育24和48小时，分别。准备博个嗜肺军团菌科比（WT或鞭毛蛋白缺乏突变体）如步骤2.1所述。 </li><li>裂解的细胞在通过加入皂苷其培养基（最终浓度:0.1％）和孵育在37℃下5分钟。 </li><li>通过移液重悬细菌和悬浮液转移到反应容器中。准备嗜肺军团菌含媒体无菌PBS系列稀释。 </li><li>条纹上BCYE琼脂板上所需的稀释液的50微升，并且在37°C孵育3天。 </li><li>视觉计算形成的殖民地。计算CFU <img alt="式（1）" src="/files/ftp_upload/55146/55146eq1.jpg" />规格化的CFU计数结果为不进行预处理，但感染的巨噬细胞，其被设置为100％。 </li></ol>

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Novartis.com Available from: <a target="_blank" href="https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause">https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause</a> (2016)</li></ol>

The authors have nothing to disclose.

细菌病原体和它们的靶细胞中的这些膜泡的影响的OMV目前正在深入研究。例如， 产气荚膜梭菌来源的OMV的诱导巨噬细胞分泌细胞因子，B淋巴细胞可以从莱姆病螺旋体的OMV被激活， 幽门螺杆菌 -released膜小泡可以在胃上皮细胞21，22，23采取行动。 嗜肺军团菌 ，细胞内病原体可诱导非典型肺炎的严重形式，也释放OMV的是能够激活肺上皮细胞和巨噬细胞16，19。在这里，我们提出了从液体培养嗜肺军团菌的OMV的小规模隔离研究的OMV在肺炎的潜在作用进行详细的协议。关键是在无菌康迪特工作离子和排除以获得纯嗜肺军团菌从其他细菌的污染来源的OMV制备。的OMV的隔离包括为了防止所得到的OMV粒料与嗜肺的污染，即使这降低了OMV产量，因为最大的OMV由该过滤步骤丢失通过0.22μm的微孔过滤步骤。 此外，我们测试了人类和小鼠巨噬细胞的隔离囊泡和感染细胞与嗜肺军团菌的响应更接近的军团菌肺炎，那里的OMV是由细菌外15日发布的LCV里面的情况。根据在孵育24小时后的体外人类巨噬细胞的感染的自由OMV量所采用的OMV剂量已被估计（在参考20描述）。对其他受体细胞的刺激或在体内实验中，其他OMV剂量可能是必要的，并且必须被建立。的嗜肺军团菌的OMV的效果分析表示前进到由雅格和施泰纳特24中描述的协议。 在这里，PMA分化的THP-1细胞作为肺泡巨噬细胞模型，由于主要人力物力有限的可用性。在加入PMA的区分单核细胞THP-1细胞为巨噬细胞样细胞25。此外，它们是嗜肺军团菌一个知名模特细胞系研究26。除了这种人类单核细胞系中，使用mBMDM细胞。 mBMDM被广泛用于嗜肺军团菌 27，28，29的影响的研究接受。利用基因敲除针对不同的TLR或其他蛋白质的可能性，使他们学习OMV效果的宝贵工具。在ORD器来降低每个实验小鼠的量，mBMDM来代替由于巨噬细胞的局限性肺泡巨噬细胞。关键的实验可能需要进行验证肺泡巨噬细胞。 除超速离心的本文所述协议来净化OMV的，它是能够进行密度梯度离心，它包括在由Chutkan 等的协议 30。这可以提高所得OMV制备的纯度，降低共纯化的蛋白聚集体，鞭毛蛋白，和LPS的量。所得到的OMV制备物的纯度可以通过透射电子显微镜或通过纳米粒子追踪分析的质量控制的补充步骤进行分析。这可以提供定量的另一种手段，超出这里提出的蛋白质测量程序。任选地，脂多糖浓度可以通过鲎变形细胞溶解物测试进行分析。如果OMV产量低，一经离心过滤器额外的浓缩步骤可以执行，这是不是在这里完成的。如果产量低于预期，OMV的被丢弃。 由于持续的努力阐明的OMV背后的生物学机制和功能的一部分，不同压力条件对OMV生产的影响可能进行测试。营养缺乏，改变培养温度，或暴露于有害物质可能对OMV分泌31产生影响。可能的应力条件在由Klimentova和Stulik 32的协议进行讨论。此外，可产生高血糖或hypovesiculating 嗜肺军团菌突变体。不同的OMV制剂然后可以在感染实验来分析与巨噬细胞，人肺组织外植体（在参考17中所述），或甚至在体内模型。另外的OMV在天然免疫信号中的作用，它们在细菌传播的影响力可以通过实验来解决。此外，各种先天免疫信号级联的影响可能通过使用鼠敲除细胞或CRISPR / Cas9击倒在人类细胞系的产生进行分析。在OMV的这个基本的研究将有助于新疫苗策略，已经存在由脑膜炎奈瑟菌 33传送乙脑的发展。 从上OMV分离和表征的协议开始，可以应用这对其它革兰氏阴性菌和其他宿主细胞;它只需要进行调整，以细菌的液体培养生长。通过Chutkan 等发表的协议。提供了来自大肠杆菌和绿脓杆菌 30产生的OMV的详细信息。培养不应以避免在裂解细菌的增加和污染蛋白和mem到达晚固定相branes。此外，用于宿主细胞的刺激OMV剂量需要根据OMV的本的过程中在体内感染的量来确定的，同时仍确保细胞毒性的低速率。以这种方式，的OMV的病理作用，它们对物种间的通信，以及宿主 - 病原体相互作用的影响可以被检查。 为了进一步研究嗜肺军团菌的OMV的肺炎中的作用，就需要有足够的产量和可比感染实验标准化OMV准备。该协议将有助于标准化分离程序，并监查研究扩展到其它的革兰氏阴性细菌和其他宿主细胞。此外，研究将在体外的知识，这可以用来延长实验在体内设置从详细受益。在未来，该协议可以扩展到从主生物材料的OMV，如血清或支气管LAV的隔离年龄液，以深入了解的OMV的生理条件下释放的成分。这将有助于确定OMV组成的关键参数，并了解在体外的OMV -生成的属性。

<html>细菌可以通过不同的机制1分泌致病因素。除了众所周知的分泌系统，革兰氏阴性细菌可以交换信息，并通过外膜囊泡（OMV的），这是小的，球体囊泡10-300纳米的粒径和具有双层膜结构传送的毒力因子。它们分泌的各种生长环境（液体培养，固体培养和生物膜），并在各生长阶段2，3。 OMV的是运输的重要手段（ 例如，蛋白质，粘附，毒素，和酶，以及对于LPS，该监查表面上找到）4。腔内货物免受蛋白水解降解，所以它能够在长距离行事，以及囊泡可在体液和远处器官5,6发现，"外部参照"&gt; 7，8。它们不仅可以识别和真核细胞9,10占用，但此外，它们能够促进细菌及其侵袭的结合到宿主细胞4。 嗜肺军团菌 （ 嗜肺军团菌 ）是一种革兰氏阴性细菌，可以释放的OMV。在人的肺，它主要感染肺泡巨噬细胞，尽管它的自然宿主是淡水变形虫11。一个嗜肺军团菌感染可引起军团病，肺炎12是一种严重的。在宿主细胞中它的块吞噬体 - 溶酶体融合。它还募集宿主细胞器，由此复制小生， 军团含液泡（LCV），形成13，14。溶酶体降解被抑制不仅效应蛋白TRAnslocation经由IV型分泌系统，也受到的OMV 15的释放。 的OMV的从细菌培养物中纯化需要分析其对受体细胞的效果。早期的研究集中于嗜肺军团菌的OMV的蛋白质含量和在囊泡上肺泡上皮细胞16，但与人肺组织移植后的研究的影响证明了嗜肺 OMV的由肺泡巨噬细胞17吸收。 作为OMV的本病原体相关分子模式（PAMP）和其他细菌抗原，它们可能对感染真核细胞的影响和调节宿主的免疫反应18。 嗜肺军团菌的OMV迅速与宿主细胞膜融合，而且，他们激活膜质TLR2 19。因为已知的是L. pneumophILA OMV的刺激巨噬细胞和上皮细胞的促炎方式16，图 17，我们分析的OMV对感染过程在人类和小鼠巨噬细胞的影响。 在这里，我们描述了嗜肺军团菌的培养的协议在液体培养通过差超速离心对分泌的OMV隔离并评估囊泡上真核宿主细胞的影响，无论是直接或以下的感染。

<table border="0" cellpadding="0" cellspacing="0" width="1938">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm petridish</td>
			<td style="width:475px;">Sarstedt AG &#38; Co KG (Nuembrecht, Germany)</td>
			<td align="right" style="width:119px;">82.1473</td>
			<td style="width:1128px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 Ti rotor</td>
			<td style="width:475px;">Beckman Coulter Incorporation (California, USA)</td>
			<td align="right">337922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACES</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">9138.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">activated charcoal</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td style="width:119px;">X865.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">agar-agar, Kobe I</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">5210.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Columbia agar with 5 % sheep blood</td>
			<td style="width:475px;">Becton Dickinson GmbH (Heidelberg, Germany)</td>
			<td align="right" style="width:119px;">254005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cuvettes</td>
			<td style="width:475px;">Sarstedt AG &#38; Co KG (Nuembrecht, Germany)</td>
			<td align="right" style="width:119px;">67.742</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ELISA (human)</td>
			<td style="width:475px;">BD OptEIA&#8482;; Becton Dickinson GmbH (Heidelberg, Germany)</td>
			<td style="width:119px;">IL-8: 555244 IL-6: 550799</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ELISA (murine)</td>
			<td style="width:475px;">DuoSet, R&#38;D (Minneapolis, USA)</td>
			<td style="width:119px;">CXCL1: DY453-05</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Fe(NO3)3x9H2O</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">5632.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum (FCS)</td>
			<td>Life Technologies GmbH (Darmstadt, Germany)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Heracell 240i CO2 incubator</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td align="right">40830469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heraeus Multifuge X3R</td>
			<td>Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td align="right">75004515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inoculation loop&#160;</td>
			<td style="width:475px;">Sarstedt AG &#38; Co KG (Nuembrecht, Germany)</td>
			<td style="width:119px;">86.1567.010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KOH</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right" style="width:119px;">6751.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L. pneumophila Corby</td>
			<td style="width:475px;">---</td>
			<td style="width:119px;">---</td>
			<td>kindly provided by Prof Dr Antje Flieger (RKI, Berlin, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L. pneumophila Corby &#916;flaA</td>
			<td style="width:475px;">---</td>
			<td style="width:119px;">---</td>
			<td>kindly provided by Prof Dr Klaus Heuner (RKI, Berlin, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-cystein</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mBMDM</td>
			<td style="width:475px;">---</td>
			<td style="width:119px;">---</td>
			<td>kindly provided by Prof Dr Markus Schnare (Philipps Univeristy Marburg, Marburg, Germany) and Prof Dr Carsten Kirschning (University Duisburg Essen, Essen, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td>Biochrom GmbH (Berlin, Germany)</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phorbol 12-myristate 13-acetate</td>
			<td style="width:475px;">Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">rotating shaker (MaxQ 6000)</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td>SHKE6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 high glucose</td>
			<td>Life Technologies GmbH (Darmstadt, Germany)</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">saponin</td>
			<td style="width:475px;">Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right" style="width:119px;">9622.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrospec 10</td>
			<td style="width:475px;">Biochrom Ltd (Cambridge, England)</td>
			<td style="width:119px;">80-2116-30</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile filter (pore size: 0.22 &#181;m)</td>
			<td style="width:475px;">Corning Incorporated (new York, USA)</td>
			<td align="right" style="width:119px;">431096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">THP-1</td>
			<td style="width:475px;">Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td>
			<td>88081201-1VL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorvall Discovery 100 SE</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">yeast extract</td>
			<td>Carl Roth GmbH &#38; Co KG (Karlsruhe, Germany)</td>
			<td align="right">2363.2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA protein assay kit</td>
			<td style="width:475px;">Thermo Fisher Scientific Germany BV &#38; Co KG (Braunschweig, Germany)</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

legionella pneumophila outer membrane vesicles: isolation and analysis of their pro-inflammatory potential on macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

实验装置准备嗜肺军团菌的OMV，并分析其对感染后图1描述了巨噬细胞的促炎性反应的影响。所制备的OMV的促炎症潜力可以在PMA分化的THP-1细胞，其在图2中所示进行分析。 THP-1细胞与IL-8和IL-6分泌的时间和剂量依赖性增加作出反应。此外，在嗜肺军团菌的OMV识别不同TLR的影响可以通过使用mBMDM从不同的遗传背景进行分析，如在图3中由CXCL1 ELISA分析。 mBMDM从OMV刺激后分泌CXCL1野生型小鼠，而mBMDM TLR2 / 4 - / -分泌显著少。研究嗜肺军团菌的OMV对细菌复制在THP-1巨噬细胞的影响，将细胞预温育与OMV的再另外感染与嗜肺军团菌 （ 图4 A）。 THP-1巨噬细胞的预刺激第一减少感染的24小时后的细菌复制，但是它会导致在CFU翻倍在稍后的时间点（48小时PI）的计数。感染的巨噬细胞的以下信号上的OMV识别Toll样受体（TLR）的影响，可以通过mBMDM进行评估，如在图4 B中。由十倍mBMDM来自WT动物OMV预孵育后细菌复制的增加，而TLR2 - / -和TRIF / MyD88的- / -细胞不显示这种增加嗜肺军团菌的复制。 <img alt="图1" src="/files/ftp_upload/55146/55146fig1.jpg" /> 图1:实验步骤。 （A）的 嗜肺军团菌科比WT从10厘米的BCYE琼脂板上被用于接种小液体培养（10毫升），其被转移到90毫升新鲜的YEB中6小时后网上平台。小体积也接种于血琼脂平板上，以检查纯度。细菌在37℃下培养，直到早期稳定期（OD 600 = 3.0-3.5）。 （B）中的液体培养物离心并无菌过滤以除去细菌。然后军团 -free上清液超离心以获得OMV粒料，其再悬浮在PBS中并超速离心一次。分离的囊泡再悬浮，检查纯度，以及该蛋白量进行定量。比例尺条为2.5厘米。 （C）的人或鼠巨噬细胞刺激与量化的OMV。细胞培养上清液可用于酶联免疫吸附，或巨噬细胞可以与嗜肺感染由CFU试验上10厘米BCYE琼脂板上以确定细菌复制。 <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </一&gt; <img alt="图2" src="/files/ftp_upload/55146/55146fig2.jpg" /> 图2:由嗜肺军团菌的OMV THP-1细胞的促炎活性。 （A）的在此，单核细胞THP-1细胞系被用作肺泡巨噬细胞的模型。 PMA分化的THP-1细胞用24和48小时，分别增加剂量嗜肺军团菌的OMV（0.01-25微克/毫升）处理。用于IL-8的ELISA中无细胞上清液。三个独立实验±SEM的平均值被示出。 THP-1细胞的反应，以尽可能少为0.01微克/毫升嗜肺军团菌的OMV与显著的IL-8的分泌，这是时间和剂量依赖性。 （B）的 嗜肺军团菌的OMV（0.1-10微克/毫升）用于刺激PMA分化的THP-1细胞。上清液孵育24和48小时后收集，且所释放的IL-6通过ELISA法在上清液中测得的。三个独立实验±SEM的平均值被示出。 THP-1细胞分泌的显著量的IL-6，即使采用的OMV的最低剂量（0.1微克/毫升）。 IL-6的分泌随着OMV剂量和延长温育时间增加。统计:Mann-Whitney检验; * P &lt;0.05和** P &lt;0.01，相比于相应0微克/毫升的OMV。经许可后转载自参考20. <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55146/55146fig3.jpg" /> 图3:巨噬细胞的促炎活性取决于TLR2 / 4。 mBMDM来自WT和TLR2 / 4 - / -小鼠用嗜肺军团菌的OMV（0.1或1微克/毫升）一起温育。 CXCL1分泌用ELISA 24和48小时后分别分析。该±三个独立实验的SEM平均值被示出。 mBMDM来自WT小鼠的回应嗜肺 OMV孵育后以剂量依赖性CXCL1分泌。 TLR2 / 4 - / -与WT相比mBMDM mBMDM分泌显著少CXCL1，这种分泌没有增加剂量依赖性。统计:Mann-Whitney检验; * P &lt;0.05，相比于相应0微克/毫升的OMV; ＃P &lt;0.05相比，同等对待WT样本。经许可后转载自参考20. <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55146/55146fig4.jpg" /> 图4:在巨噬细胞嗜肺 OMV预孵育增加细菌复制。 （A）的分化的THP-1细胞预温育与OMV的（0.1,1或10μ克/毫升）或LPS / IFN-γ（200毫微克/毫升的每个）或不进行处理控制。预孵育（20小时）后，THP-1细胞分别感染嗜肺科比WT（MOI 0.5）2，24，和48小时，。 THP-1细胞通过加入皂苷裂解，将细菌铺在BCYE琼脂板上。 CFU的是各时间点后，0微克/毫升OMV相对计算。条形代表±三次独立实验，每次在技术重复进行SEM的平均值。有在细菌摄取（2小时后的感染（PI））相比，没有预先处理的细胞没有差异。在细菌复制改建分别24和48小时后测定。 LPS / IFN-γ预处理的THP-1细胞表现出细菌负荷24小时圆周率的降低这也观察剂量依赖性地为嗜肺军团菌的OMV预先处理的细胞。在稍后的时间点（48小时圆周率），经预处理的OMV THP-1细胞表现出嗜肺重增加了一倍褶皱，而LPS / IFN-γ预处理的巨噬细胞表现出细菌负荷的进一步的降低。统计:Mann-Whitney检验; * P &lt;0.05和** P &lt;0.01，相比于相应0微克/毫升的OMV。 （B）的 mBMDM来自具有不同遗传背景的小鼠（WT，TLR2 - / - ，和TRIF / MyD88的- / - ）中预温育用0.1微克/毫升嗜肺军团菌的OMV 20小时和然后感染了鞭毛蛋白嗜肺军团菌科比（MOI 0.5），48小时的缺失突变体。 mBMDM通过加入皂苷裂解，和军团菌接种在BCYE琼脂板上。在CFU被相对于0微克/毫升的OMV计算，由实线表示。条形代表平均值±±的三个独立的实验中，每个在重复进行扫描电镜。 mBMDM来自WT小鼠显示OMV预处理后的增加嗜肺军团菌的复制。 TLR2 - / -巨噬细胞表现出显著降低<em&gt;军团复制，这是堪比TRIF / MyD88的- / - mBMDM。统计:Mann-Whitney检验; P &lt;0.05相对于WT样品。经许可后转载自参考20. <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages at JoVE.com

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

这里，我们描述嗜肺军团菌 （ 嗜肺军团菌 ）从液体培养外膜囊泡（OMV的）的纯化。然后将这些纯化的囊泡用于巨噬细胞的治疗，以分析其促炎潜力。

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 ...

legionella-pneumophila-outer-membrane-vesicles-isolation-analysis

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JoVE Journal

Immunology and Infection

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

11.1K 次观看.  Philipps-University Marburg. 这里，我们描述嗜肺军团菌 （ 嗜肺军团菌 ）从液体培养外膜囊泡（OMV的）的纯化。然后将这些纯化的囊泡用于巨噬细胞的治疗，以分析其促炎潜力。 

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

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

科研

免疫与感染

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

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. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

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 ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

巨噬细胞吞噬胸腺细胞吞噬的实验分析

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...