多米尼克·米尔斯由布卢姆斯伯里高校博士助学金（2007-2010年）的支持。笔者想感谢他们协助开发的VDC模型的OZAN Gundogdu和阿卜迪·艾尔米。

1。 IEC单层生长的特殊0.4微米的过滤器<ol><li>文化活动碳酸钙-2细胞在Dulbecco氏修改的必不可少的介质（DMEM）与10％（体积/体积）胎儿小牛血清，100 U / ml的青霉素，100μg/ ml的链霉素和1％（体积/体积）中的不必要的氨基酸补充标准组织培养孵化器在37℃下在5％CO 2和95％空气。种子4×10 5的 Caco-2每0.4微米过滤器的IEC和增长极化超过21天，每2-3天改变媒体。 </li><li>测量的TEER的单层使用伏欧姆电阻表确认极化状态。在我们的研究中，21天的TEER的Caco-2细胞生长在这0.4μm的过滤器是700-800欧姆厘米2。 </li></ol> 2。 C.制备空肠弯曲菌和细菌的共培养培养基含量<ol><li> 24小时前VDC共培养实验所需的起始点，准备一个新鲜的血琼脂平板上的C。空肠弯曲菌和孵化，在37℃微氧条件下（85％N 2，O 2 5％，10％CO 2）在变量的气氛孵化器。 </li><li>在同一时间，垫上预温育30毫升的布鲁氏杆菌肉汤中，于37℃微需氧条件下，在一个可变气氛的孵化器。 </li></ol> 3。制备和灭菌，检测前的VDC半钱伯斯<ol><li>完全沉浸VDC半室以及O型圈，并在消毒溶液中的插头。 </li><li>使用无菌的20毫升注射器，通过进气口的两个半室灭菌溶液冲洗。如果不这样做，可能会导致共培养过程中的样品污染。 </li><li>保留所有组件沉浸在1小时的灭菌水溶液。 </li><li>用新鲜无菌水冲洗所有组件，用另一个无菌的20毫升注射器冲洗进气口。 </li></ol> 4。准备细菌接种物<ol><li>收获C.半一盘菌生长的血琼脂平板准备的前一天为1毫升的预孵育布氏肉汤。这应该产生〜10 CFU /毫升。 </li><li>的细菌悬浮液的光密度测量，在600nm semiquantify细菌菌落形成单位（的CFUs）。 </li><li>调整到所需的接种量在4毫升的预培养的布鲁氏杆菌肉汤中的细菌悬浮液。 </li><li>从这个最后的细菌悬浮液，然后通过电镀适当的稀释血琼脂平板上，一式三份，然后在37℃下孵育48小时，在变量气氛培养箱量化存在于接种物中的细菌的CFUs的数目进行连续稀释。 </li></ol> 5。设置直流电压<ol><li>将VDC单位的下半部室的板凳上。适合O型圈到下半部室。 </li><li>分离一个过滤器从t携带的IEC他载波和400微升无菌PBS洗三次。将过滤器到的下半部室，确保O形圈保持在原位。 </li><li>轻轻放下的上半腔到位。两个半室后组装而成，使用环型夹夹在一起。将VDC水平在板凳上顶部，与两个开口朝上。 </li><li>添加4毫升细菌接种到心尖的半室。 </li><li>添加4毫升细胞培养液为基底的半室。 </li><li>将末尾件到两个半室上的开口。 </li></ol> 6。安装VDC气歧管内的可变大气孵化的<ol><li>将VDC到变量的气氛培养箱在靠近气歧管。 </li><li>打开连接的气体歧管的气体供给调节器。从歧管上的管连接到进气口上的基底侧的半室的直流电压。附加的GA口门管领导变量的气氛孵化到底片基底的半室的网点之一。 </li><li>关闭关闭其他出口到底片基底的半室。打开气流气歧管上，照顾打开很慢，以避免多余的气体压力，因为这可能会导致驱逐基底介质。 </li><li>宗旨是每2-5秒1气泡的气体流。定期进行检查，在实验过程中气体的流动过程中，必要时调整。 </li></ol> 7。拆卸VDC共培养后<ol><li>经过适当的共培养期间，关闭气源稳压器连接到气歧管。关闭气体流量到VDC歧管。断开气体入口，气体出口和从直流电压中的止动件。 </li><li>删除VDC从可变的气氛孵化。删除心尖和基底上清和储存在-80°C subsequent检验。 </li><li>拆下环形夹子和轻轻拆开VDC。从半室转移到无菌的6孔细胞培养皿中取出过滤器。 IECS 3次洗净，用400μl的无菌PBS。 </li><li>对于枚举相互作用的细菌总数，添加400μl的无菌PBS含有0.1％（体积/体积）的Triton X-100的IEC，并在室温下孵育20分钟以裂解的IEC。随后通过电镀适当的稀释血琼脂平板上，一式三份，然后在37℃下孵育48小时，在变量气氛培养箱量化相互作用的细菌的CFUs的数目从该细胞裂解物进行连续稀释。 </li><li>对于枚举入侵的细菌总数，在5％CO 2和95％DMEM培养液细胞培养液中含有150微克/毫升庆大霉素的IEC和在标准组织培养孵化2小时孵育在37℃下加入400微升杀灭空气中胞菌，然后按照上述步骤7.4。 </li></ol> 8。清洁VDC共培养后洗净，用无菌水和存储，为下一轮实验的直流电压。

<ol>
	<li>Mills, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+Campylobacter+jejuni+invasion+of+intestinal+epithelial+cells+under+low-oxygen+coculture+conditions+that+reflect+the+in+vivo+environment.">Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.</a> Infect. Immun. 80, 1690-1698 (2012).</li><li>Marteyn, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Shigella+virulence+in+response+to+available+oxygen+in+vivo.">Modulation of Shigella virulence in response to available oxygen in vivo.</a> Nature. 465, 355-358 (2010).</li><li>Dorrell, N., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+century+of+Campylobacter+research:+recent+advances,+new+opportunities+and+old+problems.">The second century of Campylobacter research: recent advances, new opportunities and old problems.</a> Curr. Opin. Infect. Dis. 20, 514-518 (2007).</li><li>Young, K. T., Davis, L. M., Dirita, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni:+molecular+biology+and+pathogenesis.">Campylobacter jejuni: molecular biology and pathogenesis.</a> Nat. Rev. Microbiol. 5, 665-679 (2007).</li><li>Hu, L., Kopecko, D. J., Nachamkin, I., Szymanski, C. M., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17.">Chapter 17.</a> Campylobacter. , 297-313 (2008).</li><li>Everest, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Caco-2+cells+as+a+model+for+enteric+invasion+by+Campylobacter+jejuni+and+C.+coli.">Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.</a> J. Med. Microbiol. 37, 319-325 (1992).</li><li>Konkel, M. E., Hayes, S. F., Joens, L. A., Cieplak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+internalization+and+intracellular+survival+of+Campylobacter+jejuni+in+human+epithelial+cell+cultures.">Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.</a> Microb. Pathog. 13, 357-370 (1992).</li><li>Monteville, M. R., Konkel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-facilitated+invasion+of+T84+eukaryotic+cells+by+Campylobacter+jejuni+occurs+preferentially+at+the+basolateral+cell+surface.">Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface.</a> Infect. Immun. 70, 6665-6671 (2002).</li><li>Friis, L. M., Pin, C., Pearson, B. M., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+methods+for+investigating+Campylobacter+invasion+mechanisms.">In vitro cell culture methods for investigating Campylobacter invasion mechanisms.</a> J. Microbiol. Methods. 61, 145-160 (2005).</li><li>Schuller, S., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerobic+conditions+enhance+type+III+secretion+and+adherence+of+enterohaemorrhagic+Escherichia+coli+to+polarized+human+intestinal+epithelial+cells.">Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells.</a> Environ. Microbiol. 12, 2426-2435 (2010).</li><li>Cottet, S., Corthesy-Theulaz, I., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerophilic+conditions+permit+to+mimic+in+vitro+events+occurring+during+in+vivo+Helicobacter+pylori+infection+and+to+identify+Rho/Ras-associated+proteins+in+cellular+signaling.">Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.</a> J. Biol. Chem. 277, 33978-33986 (2002).</li></ol>

我们什么都没有透露。

研究细菌病原体与宿主细胞的相互作用是在体外细胞培养的方法，使用广泛采用的一种技术，在许多研究实验室。然而，由于这样的细胞培养实验是在有氧条件下进行的，这些在体外模型中，可能无法精确地表示在体内环境，使宿主病原体相互作用发生。 在体外感染模型被广泛使用的是特别不合适的学习微需氧C.菌相比其他肠道致病菌，兼性厌氧菌。在文献中有?...

与宿主细胞的细菌性病原体的相互作用已被广泛研究，利用体外细胞培养方法。使用这样的细胞培养实验中，细菌宿主细胞的粘附性，鉴定的受体宿主细胞，宿主细胞的信号转导途径和细菌入侵宿主细胞都被详细研究，导致在许多重要的观测。然而，这样的细胞培养实验是在有氧条件下，可能不具有代表性的体内环境。 在体外模型用来研究胃肠道感染是一个主要的限制，文化条件，包括高氧含量普遍青睐真核细胞的存活。然而，在肠腔内的条件下将几乎是厌氧的。在一个非常低的氧环境下的肠道病原体表达的毒力基因的表达在有氧条件下的变化2。因此，获得的数据，使用标准的细胞培养模型M可能给出不准确的指示细菌与宿主细胞的相互作用。 空肠弯曲菌是细菌性急性胃肠炎全球领先的病原体，症状范围从轻度腹泻到严重的炎症性肠炎。 C.大多数空肠弯曲菌感染导致并发症的胃肠炎，但是C.空肠弯曲菌是最常见的周围神经病变，包括格林-巴利综合征（GBS）的传染性病原体。据估计，在英国，有超过50万的情况下，引起肠炎C.菌感染，每年对英国经济的预测成本5.8亿英镑。在发展中世界，C.菌是儿童死亡的首要原因。尽管无疑是十分重要的弯曲杆菌感染和几十年的研究，包括彻底的基于基因组学的分析，C.菌的发病机制仍是不佳understOOD，对比其他肠道致病菌如沙门氏菌 ， 大肠杆菌 ， 痢疾杆菌 ， 霍乱弧菌 。一个方便的小动物模型的缺乏是其中一个主要原因，这3。此外，广泛应用于体外感染模型更不适用于研究微需氧C.菌比其他肠道致病菌，兼性厌氧菌。虽然C。菌侵入的病原体，机制C.确认为菌侵入肠上皮细胞（IECS）目前仍不清楚。C. 4,5 空肠弯曲菌侵袭已被证明是依赖于以微丝，微管，两者的组合或两者都不是5。在这方面的混乱，最有可能是由于在体外试验条件下的使用不恰当的。 已使用的许多不同的细胞培养实验，调查C的相互作用菌与宿主细胞。 Caco-2细胞，INT 407 7，T84 8芯线都被用于研究不同C.黏附和侵袭能力菌菌株。不过，该水平的细菌粘附和入侵C.空肠弯曲菌与IECS是大大低于其他肠道病原体9。共培养C.空肠弯曲菌与IECS通常是附近的大气中的氧水平的条件下，在CO 2培养箱中进行，所需的生存的IEC C。空肠弯曲菌基因表达会有很大的不同，在低氧气的环境相比，大气中的氧气条件下肠腔。 已开发的垂直扩散商会系统（VDC）的使用，它允许根据不同的介质和气体条件1,10,11细菌与宿主细胞的共培养。该系统模拟的条件在人体肠道中的细菌将被取消从血液中的氧会被提供一个非常低的氧，而组织的性别条件。有极性的IEC单层生长在特殊的0.4μm的过滤器被放置成一个直流电压，创建心尖和基底的隔室，与细菌的发酵液和细胞培养基中（ 图1），并分别填充。的直流电压被放入变量的气氛含有85％N 2，5％O 2，和10％的CO 2培养箱在37℃下，较下的最佳条件为空肠弯曲菌 。心尖室敞开和变量气氛培养箱内暴露于微氧气氛，而密封的基底外侧隔室与氧气的供给恒定给药5％CO 2/95％O 2的混合气体与出口管防止压力积累。获确认通过监测跨膜元素，Caco-2细胞的生存和在这些条件下的单层的完整性跨单层超过24小时ctrical电阻（TEER），以证明生存的Caco-2细胞单层和物理分离的心尖和基底低氧条件下在心尖的隔室的隔室。单层TEER在变量的气氛孵化器（微氧条件）和标准的细胞培养CO 2培养箱（有氧条件下）保持在VDC的没有表现出显着性差异，表明细胞单层的完整性不同大气条件下1。微氧条件下，紧密连接仍然存在occludin的染色模式类似于细胞维持在有氧条件下1单元格边框之间均匀分布。 C.的相互作用空肠弯曲菌与Caco-2细胞和T84细胞在VDC进行了研究，通过评估细菌相互作用（粘附和侵袭）和侵袭。两种不同的C。菌野生型笔直NS 1。C.空肠弯曲菌 11168H是一个hypermotile的的原始序列菌株NCTC11168的衍生物。 11168H应变显示在一个小鸡定植模型定植水平高得多，因此被认为是一个合适的菌株用于宿主 - 病原体相互作用的研究。 81-176是人类的隔离是最侵入性的广泛的研究的实验室菌株之一。C.空肠弯曲菌菌株中加入微需氧或好氧条件下向根尖隔室的一个直流电压。我们观察到互动和侵袭率较高C.录菌微氧条件下1。 C.增加菌相互作用不是由于细菌数量增加，微氧条件下1。在心尖的隔室中的VDC的低氧环境中增强了细菌宿主的相互作用，并指示此数据支持了我们的假设，C.侵入性的性质的空肠弯曲菌是增量在这些条件下的缓解。这是首次报告使用的VDC模型研究侵袭性细菌病原体，并进行体外感染实验条件下，更加紧密地模仿体内的情况突出的意义。 VDC模型可以用来研究许多不同的细菌宿主 - 病原体相互作用。

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Speciality vertical diffusion system for use with Snapwell inserts</td>
			<td>Harvard Apparatus</td>
			<td>66-0001</td>
			<td>Manifold &#38; six Snapwell chambers</td>
		</tr>
		<tr>
			<td>Caps</td>
			<td>Harvard Apparatus</td>
			<td>66-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>Harvard Apparatus</td>
			<td>66-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Harvard Apparatus</td>
			<td>66-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Snapwell filters (pore size 0.4 &#956;m)</td>
			<td>Corning Costar</td>
			<td>3407</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS-2 Volt-Ohm resistance meter</td>
			<td>Millipore</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>WPA Lightwave II spectrophotometer</td>
			<td>Biochrom</td>
			<td>80-3003-72</td>
			<td></td>
		</tr>
	</tbody>
</table>

enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model

与宿主细胞的细菌性病原体的相互作用已被广泛研究，利用体外细胞培养方法。然而，由于这样的细胞培养实验是在有氧条件下进行的，这些在体外模型中，可能无法精确地表示在体内环境，使宿主病原体相互作用发生。我们已经开发出了不同的介质和气体条件下，允许细菌与宿主细胞的共培养感染的体外模型。厅（VDC）的垂直扩散模型模拟人体肠道的条件下，细菌会在那里将提供与从血液中的氧的条件下，是非常低的氧，而组织。配售极化肠上皮细胞（IEC）单层生长成VDC在Snapwell插入心尖和基底创建单独的车厢。基底外侧的隔室充满了细胞培养液中，密封，灌注与氧瓦特休斯特根尖车厢充满了肉汤，保持开放和微氧条件下培养。双方的Caco-2和T84的IEC可以维持在直流电压，在这些条件下，对​​细胞的存活或单层的完整性没有任何明显的不利影响。共培养实验进行不同C.空肠弯曲菌的野生型菌株和不同的IEC线VDC模型再现性与微需氧条件下在心尖室产生相互作用的数量（近10倍）的增加和细胞内细菌（近100倍）相比，有氧培养条件1。 VDC模型中创建的环境更加紧密地模仿环境C.遇到在人体肠道菌和突出表演体外感染实验条件下，更加紧密地模仿在体内现实中的重要性。我们建议，使用的VDC模型将允许下注的相互作用的新诠释WEEN细菌病原体和宿主细胞。

共培养实验用C.空肠弯曲菌的野生型菌株和在VDC与微需氧条件下在心尖室模型的IEC已经证实，在相互作用的数量增加（几乎10倍）和细胞内（近100倍）相比，细菌的需氧培养条件下，在一个时间依赖的方式1。这一观察结果是再现的使用两个不同的C。空肠弯曲菌野生型菌株（11168H 81-176）和两个不同的IEC线（Caco-2细胞和T84），突出VDC模型1的有效性。 <p class="jove_content"...

Watch this Scientific Journal Video about Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model at JoVE.com

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

在有氧条件下进行细胞培养实验调查细菌的粘附和侵袭，通常是不具代表性的<em&gt;在体内</em&gt;环境。 A垂直扩散室模型允许人类病原体的相互作用研究<em&gt;空肠弯曲菌</em与肠上皮细胞在更<em&gt;在体内</em&gt;如条件，从而增强细菌的侵袭。

肠道细菌侵入肠上皮细胞<em&gt;体外</em&gt;使用垂直扩散室模型，显着增强

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

enteric-bacterial-invasion-intestinal-epithelial-cells-vitro-is

Research

JoVE Journal

Immunology and Infection

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

13.7K 次观看.  London School of Hygiene & Tropical Medicine. 在有氧条件下进行细胞培养实验调查细菌的粘附和侵袭，通常是不具代表性的在体内环境。 A垂直扩散室模型允许人类病原体的相互作用研究空肠弯曲菌

视频: Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

科研

免疫与感染

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

使用人类肠道上皮细胞和BALB / C小鼠巨噬细胞的容忍脂肪酸和霍乱毒素浓度的测定

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...