The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.

注意:在整个实验中，巨噬细胞，在5％CO 2的强制空气培养箱中在37℃下保温，并仅用于实验操作，其在II类生物安全柜进行取出的孵化。与L.工作菌等细菌是根据生物安全2级法规。 1.细胞的制备及细菌感染（1日和2） <ol><li> 1天<ol><li>种子2.0×10 7个骨髓衍生巨噬细胞（BMDM）上在30毫升BMDM +青霉素-链霉素介质（ 表2）145毫米的菜。种子3板为每个细菌菌株进行分析。在一个5％CO 2的强制空气培养箱中在37℃孵育过夜。 </li><li>启动野生型（WT）L.的过夜细菌培养在标准培养箱单核细胞株10403S在10ml脑心脏浸液（BHI）培养基中，在30℃下。地方培养管倾斜无晃动。注意:这些生长条件上调鞭毛基因的表达，从而促进有效的感染。 </li></ol></li><li>第2天<ol><li>预暖BMDM介质（不含青霉素-链霉素抗生素）（ 表2）和磷酸盐缓冲盐水（PBS）在37℃。 </li><li>用25毫升预热的PBS洗单层巨噬细胞两次以去除抗生素。加入30毫升的新鲜BMDM网上平台。 </li><li>通过在&gt; 14000 xg离心离心细菌1分钟，弃去上清，并通过移液在1.5ml PBS中轻轻重悬细菌洗涤1.5毫升PBS中过夜的细菌培养物。重复两次。 </li><li>每个感染巨噬细胞板用0.5毫升野生型L的洗过夜培养菌等 。如果使用多个板块，除了感染每盘15分钟。该时间间隔将使得在感染结束时分别收获每个板。 注:感染复数（MOI），是通过镀覆连续稀释测定后在37℃下板中的24小时温育用于感染上BHI琼脂平板上，计数克隆形成单位（CFU）的细菌培养的第使用0.5毫升WT L的菌菌株在〜100的MOI 10403S的结果（每巨噬细胞100细菌CFU）。当分析细胞内生长缺陷细菌突变体，更高的MOI应予以考虑。 </li><li>以下在37℃下0.5小时温育后，洗涤被感染的细胞两次用PBS以除去未附着的细菌，并添加30毫升预热BMDM介质。细菌附着接下来将0.5小时期间内化。 </li><li>在1小时感染后，添加30微升庆大霉素（1:1000，以达到50微克/ ml的终浓度），以杀死细胞外细菌。 </li><li>通过将过滤器头与一个真空出口的收集液烧瓶组装过滤装置。然后放置在过滤头的过滤器（0.45微米），接着气缸漏斗并用金属C安全的不同部分灯。准备冰冷的无RNA酶的水。 </li><li>在如下6小时感染后，收获细菌感染的细胞。个别对待每一个板块。 注:通常情况下，L。单核细胞增生完成1日志增长的过程中巨噬细胞感染了6个小时。长时间孵育可能导致细菌过度生长和巨噬细胞的细胞死亡，而较短的潜伏期，可能会导致相当低细菌负荷。在MOI和感染的时间可以被修改以达到最佳的条件。 <ol><li>洗涤感染的细胞用PBS一次。加入20毫升冰冷的无RNA酶的水以裂解细胞巨噬细胞的细胞。使用细胞刮，刮关闭单元板很快，但仔细。 </li><li>收集裂解的细胞到50ml锥形管中。涡旋30秒。离心机在800×g离心在4℃下3分钟。 </li><li>通过使用真空系统的过滤装置通过上清液。使用镊子，滚动过滤器，并迅速转移到一个15毫升的CONICAL管。用在液氮中的过滤器单元冻结管。 </li><li>组装过滤装置与用于下一个样品一个新的过滤器，如在1.2.7中描述。 </li><li>在-80℃下为第二天存储冻结滤波器。或者，直接进行核酸提取。 </li></ol></li></ol></li></ol> 2.核酸提取（3天） 注意:在通风橱中进行用苯酚和氯仿溶液的所有操作。 <ol><li>制备1:酸化苯酚1混合:氯仿，400微升各样品。混在分开的管中，然后通过抽吸撤离得到的水层。加入40微升10％SDS的。 </li><li>在冰上解冻含有过滤管，让他们感冒。到各含有过滤管添加650微升乙酸乙酯- EDTA（AE）缓冲液（ 表2）。 </li><li>尽可能快地执行以下步骤。 <ol><li>大力涡包含过滤器，这样管该过滤器搅乳到管的外周和缓冲充分洗涤该过滤器。始终放置回冰上保持管冷。注意:可能需要额外地涡管而反转为充分洗菌落滤波器。 </li><li>重复所有管。它可能是到降速不久悬浮液（在120 xg离心1分钟）在过程的结束时是有用的。 </li></ol></li><li>含细菌缓冲器转移至含有SDS和苯酚/氯仿混合物（如在2.1制备）1.5ml微量离心管中。重复所有管。可能有必要再次旋转该含过滤管（在120 xg离心1分钟），以从过滤器得到的残余缓冲液。 </li><li>将所有在多管涡旋器1.5毫升管，并涡旋全速10分钟。 </li><li>孵育在65℃的加热块的管10分钟。离心以最大速度（&gt; 14000 xg离心）5分钟。 </li><li>从各t传送，水层（约400微升）UBE到含有40微升3M乙酸钠（pH 5.2）和1.0毫升100％乙醇的新的1.5毫升管。彻底涡每管。 注意:糖原可以在这个步骤中加入，以改善沉淀的沉淀的可视化。 </li><li>孵育样品在-80℃下1小时。可替代地，在-20孵育样品℃过夜。然后，离心将样品在4℃下以最大速度（&gt; 14000 XG）20分钟。 </li><li>小心吸断从每个管的乙醇（上层）。 500微升冷的70％乙醇添加到每个样品和涡彻底。离心机在4℃下以最大速度（&gt; 1 4000 xg离心）20分钟。 </li><li>小心吸断从每个管的乙醇。干燥该样品使用真空蒸发器无热约2分钟。不要过度干燥的样品。 </li><li> 25μl的无RNA酶的水添加到每个样品。在室温下孵育20分钟。仔细涡和降速。 </li><li>测量在T RNA浓度他样本使用微量紫外可见分光光度计。期望以提取约0.5 - 1微克的每板核酸。 注:技术重复可以在此阶段进行组合。 </li></ol> 3. DNase处理<ol><li>根据在一个离心管的表1设置了反应。孵育在37℃下45分钟。添加450微升不含RNA酶的水和500μl的苯酚-氯仿- IAA混合物（ 表2），分离相的通过以最大速度（&gt; 14000 XG）离心2分钟。 </li><li>将水层转移到新管中，并通过2分钟离心以最大速度（&gt; 14000 XG）加入500μl氯仿IAA混合物（ 表2），涡旋并分离相。 </li><li>将水层转移到新管中，并加入1毫升乙醇和50微升3M乙酸钠（pH 5.2）。涡流。 注意:糖原可以在这个步骤中加入，以改善沉淀的沉淀的可视化。 </li><lI&gt;孵育在-80℃下1小时。可替代地，在-20孵育样品℃过夜。离心机在4℃下在最大速度20分钟。 </li><li>小心吸断从每个管的乙醇。 500微升冷的70％乙醇添加到每个样品和涡彻底。离心机在4℃下以最大速度20分钟。小心吸断从每个管的乙醇。 </li><li>干燥该样品使用真空蒸发器无热约2分钟。不要过度干燥的样品。 </li><li>加入12微升无RNase水，孵育在室温下，旋涡和降速2分钟。该样品中含有纯化的RNA，让他们在冰上。可替换地，溶解RNA的RNA酶的游离H 2 O与0.1毫摩尔EDTA或TE缓冲液（10mM的Tris，1mM EDTA）中。 </li><li>测量使用微量紫外可见分光光度计RNA的浓度。期待〜100纳克每样RNA（如果技术复制组合）。 注:根据本协议RNA提取是适合初学者通过多种技术受体基因的转录分析。我们通常采用RT-qPCR的分析来研究L.巨噬细胞1-4的感染过程中单核细胞的基因转录。 </li></ol>

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The authors have nothing to disclose.

这里描述的协议代表了从属细菌RNA分离的优化方法细菌单核细胞巨噬细胞在细胞内不断增长。这个协议是基于细胞差动裂解，并包括用于细菌RNA富集两个主要步骤:使用离心和过滤细菌的快速采集巨噬细胞的细胞核沉淀。这些步骤之后是一个标准的RNA提取步骤。虽然这个协议描述李斯特菌RNA的纯化，它可以很容易地修改为其它的细菌病原体。虽然方法主要针对RNA进行转录分析的纯化，...

细胞内细菌病原体─引起传染病和能生长和再现人类宿主细胞内的细菌─是全世界主要的健康关注5。入侵和哺乳动物细胞内复制，细胞内病原体已取得成熟的致病机制和因素。虽然这些机制是引起疾病的能力根本，我们知之甚少的监管和动态。因为在液体培养基中生长的细菌的基因表达分布不反映宿主细胞内的实际的环境中，人们越来越需要转录在它们的细胞内的壁龛生长的细菌的分析。这种分析将使主机触发特异性细菌适应的解密，并将有助于确定治疗方案设计新的目标。细胞内生长的细菌的转录组分析是非常具有挑战性，因为哺乳动物RNA多于由细菌RNA至少十倍。在这个手稿中，我们描述的实验方法，从单核细胞增生李斯特菌小鼠巨噬细胞中分离出不断增长的细菌RNA。提取的RNA可用于研究细胞内的适应和致病菌转录分析的各种技术，如RT-PCR，RNA测序，微阵列和其它基于杂交的技术的毒力机制。 李斯特菌是李斯特菌在人体中的病原体，临床表现主要对象是免疫功能低下者，老人和孕妇6的疾病。它 是，侵入一个宽已在宿主-病原体的相互作用被使用了几十年作为模型研究7哺乳动物细胞的阵列的革兰氏阳性兼性细胞内病原体。在侵袭，它最初驻留在液泡或吞噬体（在吞噬细胞的情况下），从它必须转义入T他宿主细胞胞质溶胶中，以便进行复制。几个毒力因子已经显示介导的逃生过程中，主要是孔隙形成溶血素，李斯特O（LLO）和两个附加的磷脂8。在胞质溶胶中的细菌使用主机肌动蛋白聚合机械推动自己对小区内的肌动蛋白丝，并从细胞扩散到细胞（ 图1）。 L.所有主要毒力因子参与入侵，细胞内生存和复制的单核细胞 ，都是由主控毒力调节转录激活，经皮射频消融8-10。 在过去十年中，许多研究由我们进行的和其他人已经成功地应用于宿主细胞2,11-15内部为胞内生长的细菌的转录组分析的方法。两种主要的方法用于细菌RNA从其基于主机的RNA分离:1）细菌RNA的选择性富集和2）RNA分离由迪菲rential细胞裂解。第一种方法依赖于（使用市售的试剂盒为例）的总RNA提取物消减杂交哺乳动物RNA分子或细菌转录序列（SCOTS）11选择性捕获。第二种方法依赖于细菌和宿主细胞中，其中在细菌细胞中保持完整的宿主细胞裂解的差分裂解。细菌细胞，然后从宿主细胞裂解物中分离，通常是通过离心，并且将RNA是使用标准技术提取。使用这种方法的主要问题是，连同完好无损的细菌，宿主细胞细胞核也分离，从而将RNA制剂仍含有哺乳动物RNA。克服这个问题的一种方式是完整的细菌使用差速离心的宿主细胞的细胞核中分离出来，虽然这过程通常需要时间提高在基因表达谱变化的提取过程中的关注。在本文中，我们提出了一个改进的和快速的巴cteria RNA提取协议，这是基于对细胞差动裂解的方法。首先，L.巨噬细胞感染单核细胞的细胞裂解用冷水。接着，巨噬细胞的核是由一个简短离心除去完整细菌迅速收集在过滤器上，从该RNA被使用的细菌核酸的热酚-SDS提取隔离。

<table>
	<tbody>
		<tr>
			<td rowspan="2">Listeria monocytogenes 10403S</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">20</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Bone marrow derived macrophages prepared from C57B/6 female mice</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">21</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>H2O, RNAse free</td>
			<td>Thermo Scientific</td>
			<td>10977-015</td>
			<td>DEPC-treated water can be used</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965039</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-088</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol&#160;&#160;</td>
			<td>Gibco</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion (BHI)</td>
			<td>Merckmillipore</td>
			<td>1104930500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol saturated pH 4.3</td>
			<td>Fisher</td>
			<td>BP1751I-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iso-amyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>W205702</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>W302406</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Fermentas,</td>
			<td>EN0521</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS 10%</td>
			<td>Sigma-Aldrich</td>
			<td>L4522</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck Millipore</td>
			<td>1070174000</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C, 5% CO2 forced-air incubator</td>
			<td>Thermo Scientific</td>
			<td>Model 3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Nunc</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Kontes glass holder for 45 mm filters</td>
			<td>Fisher</td>
			<td>K953755-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>MF-Millipore filters 45 mm, 0.45 &#181;m</td>
			<td>Merck Millipore</td>
			<td>HAWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac system</td>
			<td>Thermo Scientific</td>
			<td>SPD131DDA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>Model G560E</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>145 mm cell culture dishes</td>
			<td>Greiner</td>
			<td>639 160</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml tubes, RNase-free</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176;C incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>65 &#176;C heat block</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 &#176;C table centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes, 25 ml</td>
			<td>Greiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50 ml</td>
			<td>Greiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

rna purification from intracellularly grown listeria monocytogenes in macrophage cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

该模型系统示于图1，并包括感染L.巨噬细胞菌等细菌，这在复制巨噬细胞胞浆图2表示实验方案。 图3表示WT L.期间的毒力基因等RT-qPCR的分析典型结果单核细胞巨噬细胞的增长相比，在丰富的实验室中BHI的增长。结果显示的L的两个主要毒力因子的转录水平单核细胞增生;溶血素编码LLO毒素和Acta...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

rna-purification-from-intracellularly-grown-listeria-monocytogenes

Research

JoVE Journal

Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

9.1K 次观看.  Tel Aviv University. Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

视频: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

RNA纯化在细胞内生长

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共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

测量细菌感染小鼠和免疫反应李斯特菌

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

病原液泡的分离纯化

口头传播的

骨髓衍生的巨噬细胞产

成像InlC的分泌由细菌病原体调查感染蜂窝

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

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

实验性感染