Name: using a bacterial pathogen to probe for cellular and organismic-level host responses
Uploaded: 2019-02-22T12:30:00
Description: Watch this Scientific Journal Video about Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses at JoVE.com

根据所有适当的政府关于美国国立卫生研究院实验动物护理和使用的准则, 对老鼠进行了人道的治疗, 迈阿密大学动物护理机构批准了它们的使用和使用委员会 (议定书 16-053)。
1. 准备感染细胞
<ol><li>在 dmem 组织培养基中传播小鼠巨噬细胞样细胞系 raw 264.7, 辅以10% 的胎儿小牛血清 (以下简称 dmemce/fcs), 使用125ml 瓶和组织培养孵化器保持在 37°c% co2.</li>
	<li>感染前一天, ?...

<ol>
	<li>Freitag, N. E., Port, G. C., Miner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+-+from+saprophyte+to+intracellular+pathogen.">Listeria monocytogenes - from saprophyte to intracellular pathogen.</a> Nature Review Microbiology. 7 (9), 623-628 (2009).</li><li>Ray, K., Marteyn, B., Sansonetti, P. J., Tang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+on+the+inside:+the+intracellular+lifestyle+of+cytosolic+bacteria.">Life on the inside: the intracellular lifestyle of cytosolic bacteria.</a> Nature Review Microbiology. 7 (5), 333-340 (2009).</li><li>Miner, M. D., Port, G. C., Freitag, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+impact+of+mutational+activation+on+the+Listeria+monocytogenes+central+virulence+regulator+PrfA.">Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA.</a> Microbiology. 154, 3579-3589 (2008).</li><li>McCormack, R., 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=Perforin-2+Protects+Host+Cells+and+Mice+by+Restricting+the+Vacuole+to+Cytosol+Transitioning+of+a+Bacterial+Pathogen.">Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen.</a> Infection and Immunity. 84, 1083-1091 (2016).</li><li>Gregory, S. 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=Complementary+adhesion+molecules+promote+neutrophil-Kupffer+cell+interaction+and+the+elimination+of+bacteria+taken+up+by+the+liver.">Complementary adhesion molecules promote neutrophil-Kupffer cell interaction and the elimination of bacteria taken up by the liver.</a> Journal of Immunology. 168, 308-315 (2002).</li><li>Shi, 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=Monocyte+trafficking+to+hepatic+sites+of+bacterial+infection+is+chemokine+independent+and+directed+by+focal+intercellular+adhesion+molecule-1+expression.">Monocyte trafficking to hepatic sites of bacterial infection is chemokine independent and directed by focal intercellular adhesion molecule-1 expression.</a> Journal of Immunology. 184, 6266-6274 (2010).</li><li>Pitts, M. G., Combs, T. A., D'Orazio, S. E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+from+Both+Susceptible+and+Resistant+Mice+Efficiently+Kill+Opsonized+Listeria+monocytogenes.">Neutrophils from Both Susceptible and Resistant Mice Efficiently Kill Opsonized Listeria monocytogenes.</a> Infection and Immunity. 86, 00085 (2018).</li><li>Carr, K. D., 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=Specific+depletion+reveals+a+novel+role+for+neutrophil-mediated+protection+in+the+liver+during+Listeria+monocytogenes+infection.">Specific depletion reveals a novel role for neutrophil-mediated protection in the liver during Listeria monocytogenes infection.</a> European Journal of Immunology. 41 (9), 2666-2676 (2011).</li><li>Bl&#233;riot, 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=Liver-resident+macrophage+necroptosis+orchestrates+type+1+microbicidal+inflammation+and+type-2-mediated+tissue+repair+during+bacterial+infection.">Liver-resident macrophage necroptosis orchestrates type 1 microbicidal inflammation and type-2-mediated tissue repair during bacterial infection.</a> Immunity. 42 (1), 145-158 (2015).</li><li>Jones, G. S., D'Orazio, S. E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+are+the+predominant+cell+type+associated+with+Listeria+monocytogenes+in+the+gut,+but+they+do+not+serve+as+an+intracellular+growth+niche.">Monocytes are the predominant cell type associated with Listeria monocytogenes in the gut, but they do not serve as an intracellular growth niche.</a> Journal of Immunology. 198, 2796-2804 (2017).</li><li>Agaisse, 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=Genome-wide+RNAi+screen+for+host+factors+required+for+intracellular+bacterial+infection.">Genome-wide RNAi screen for host factors required for intracellular bacterial infection.</a> Science. 309, 1248-1251 (2005).</li><li>Cheng, L. W., 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=Use+of+RNA+interference+in+Drosophila+S2+cells+to+identify+host+pathways+controlling+compartmentalization+of+an+intracellular+pathogen.">Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen.</a> Proceedings of the National Academy of Science U S A. 102, 13646-13651 (2005).</li><li>Singh, R., Jamieson, A., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GILT+is+a+critical+host+factor+for+Listeria+monocytogenes+infection.">GILT is a critical host factor for Listeria monocytogenes infection.</a> Nature. 455, 1244-1247 (2008).</li><li>Burrack, L. S., Harper, J. W., Higgins, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation+of+vacuolar+maturation+promotes+listeriolysin+O-independent+vacuolar+escape+during+Listeria+monocytogenes+infection+of+human+cells.">Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells.</a> Cellular Microbiology. 11, 1382-1398 (2009).</li><li>Shrestha, N., 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=The+host-encoded+Heme+Regulated+Inhibitor+(HRI)+facilitates+virulence-associated+activities+of+bacterial+pathogens.">The host-encoded Heme Regulated Inhibitor (HRI) facilitates virulence-associated activities of bacterial pathogens.</a> PLoS One. 8, 68754 (2013).</li><li>Bahnan, 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+eIF2&#945;+Kinase+Heme-Regulated+Inhibitor+Protects+the+Host+from+Infection+by+Regulating+Intracellular+Pathogen+Trafficking.">The eIF2&#945; Kinase Heme-Regulated Inhibitor Protects the Host from Infection by Regulating Intracellular Pathogen Trafficking.</a> Infection and Immunity. 20, 00707 (2018).</li><li>Kortebi, M., 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=Listeria+monocytogenes+switches+from+dissemination+to+persistence+by+adopting+a+vacuolar+lifestyle+in+epithelial+cells.">Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.</a> PLoS Pathogen. 13, 1006734 (2017).</li><li>VanCleave, T. T., Pulsifer, A. R., Connor, M. G., Warawa, J. M., Lawrenz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Gentamicin+Concentration+and+Exposure+Time+on+Intracellular+Yersinia+pestis.">Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis.</a> Frontiers in Cellular and Infection Microbiology. 7, 505 (2017).</li></ol>

由于其快速和持续的细胞感染计划, lm是一个理想的病原体, 探索细胞活动, 影响感染。已经确定了一些对lm细胞感染 11、12、13、14的积极或消极影响的宿主因素。在我们的实验室中表征的两个这样的宿主因子, 即 Perforin-2 和 heme 管制抑制剂 (p2 和 hi), 调节了 lm感染过程的显著特征。?...

基于感染的实验模型具有内在的挑战性, 因为它们依赖于宿主和病原体的起始状态条件、各种病原体感染策略以及难以归因病原和宿主驱动的过程基于结果。单核细胞增生李斯特菌(lm) 由于其遗传和微生物的可追踪性、快速和持续的细胞感染策略以及相对清晰的特性, 已成为一种理想的病原体, 可以探测宿主防御反应。其纤维素和有机水平的感染表型之间的关系。lm的细胞感染通过四个不同的阶段1: (i) 细胞入侵, 结束与lm被封闭在一个液泡内;(二) 以lm为导向溶解液泡膜, 并将lm 释放到细胞溶胶中;(iii) 组织内的复制;(iv) 质膜的物理渗透, 导致直接相邻的细胞 (如上皮细胞) 的感染, 或在单生细胞中, 将lm释放到细胞外环境中。每个阶段都是由特定的 lm编码因素 (称为 "毒力因素") 推动的, 这些因素一旦被删除, 会导致细胞和动物模型的感染缺陷。这种一般的感染策略是由一些所谓的 "细胞质" 病原体2独立进化而来的。
菌落形成单元 (cfu) 检测被广泛用于评估体外 (即细胞) 和体内 (即有机) 感染结果。除了高灵敏度, 特别是对体内感染, cfu 检测提供了一个明确的读数病原体入侵和细胞内存活/增殖。cfu 检测已被广泛用于分析影响感染的lm和宿主细胞决定因素。尽管这些先前的研究提供了丰富的信息, 以分析细胞入侵和结肠内复制, cfu 的检测, 据我们所知, 还没有被用来跟踪第四阶段的lm感染过程: 细胞逃逸。在这里, 我们描述了相对简单的方法, 如何监测细胞逃逸 (以下简称 "出现") 可以通过 cfu 分析 (以及流式细胞术), 并展示了如何病理和宿主编码的因素如何调节这个阶段的lm感染周期。对细胞lm感染周期的终末期进行分析, 可以确定更多的病原体和宿主细胞感染特有的因素和活动。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:128px;">Gibco&#160;</td>
			<td style="width:119px;">A1049201</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ArC Amine Reactive Compensation Beads&#160;</td>
			<td style="width:128px;">Life Technologies</td>
			<td style="width:119px;">A10346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BHI (Brain Heart Infusion) broth</td>
			<td>EMD Milipore</td>
			<td>110493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Strainer, 70 &#181;m</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;">10199-656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:128px;">Roche</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM media&#160;</td>
			<td style="width:128px;">Gibco&#160;</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS tubes&#160;</td>
			<td style="width:128px;">BD Falcon</td>
			<td style="width:119px;">352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS - Heat Inactivated&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#8217; Balanced Salt solution</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>H6648-6X500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB agar</td>
			<td style="width:128px;">Grow Cells MS</td>
			<td style="width:119px;">MBPE-4040</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Yellow Dead Cell Stain kit&#160;</td>
			<td style="width:128px;">Life Technologies</td>
			<td style="width:119px;">L349S9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rhodamine Phalloidin&#160;</td>
			<td style="width:128px;">Thermo Fischer</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SP6800 Spectral Analyzer</td>
			<td style="width:128px;">Sony</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe 28G 1/2&#34; 1cc</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">329461</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TPP Tissue Culture 48 Well Plates&#160;</td>
			<td style="width:128px;">MIDSCI</td>
			<td>TP92048</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TPP Tissue Culture 6 Well Plates&#160;</td>
			<td style="width:128px;">MIDSCI</td>
			<td>TP92406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp eBeads</td>
			<td style="width:128px;">eBioscience</td>
			<td style="width:119px;">01-2222-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antigen</td>
			<td style="width:128px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11b&#160;</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = PE Cy5, Dilution = 1/100, Clone = M1/70</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD11c</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = AF 647, Dilution = 1/100, Clone = N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD45</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = APC Cy7, Dilution = 1/100, Clone = 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F4/80</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = PE, Dilution = 1/100, Clone = BM8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Live/Dead</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = AmCyN, Dilution = 1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ly6C</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = PacBlue, Dilution = 1/200, Clone = HK1.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MHC II</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = AF 700, Dilution = 1/200, Clone = M5/114.15.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NK 1.1</td>
			<td style="width:128px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Flurochrome = BV 605, Dilution = 1/100, Clone = PK136</td>
		</tr>
	</tbody>
</table>

using a bacterial pathogen to probe for cellular and organismic-level host responses

细菌病原体有多种策略, 一旦在真核细胞中存活和增殖。所谓的 "细胞增生李斯特菌" 病原体(单核细胞增生李斯特菌、志贺氏菌、假克氏菌、杜拉伦斯法郎和环结节) 可进入受感染的细胞溶胶。物理和酶降解的主要空泡膜。一旦进入细胞溶胶, 这些病原体既会增殖, 又会产生足够的机械力, 穿透宿主细胞的质膜, 从而感染新的细胞。在这里, 我们展示了 l . 单核细胞基因 (lm) 细胞感染周期的这一结束步骤如何通过成菌性系和流式细胞术来量化, 并举例说明病因和宿主编码因素是如何影响这一点的。过程。我们还显示了体外感染的培养细胞的 lm 感染动力学与体内感染小鼠的肝细胞的lm感染动力学的密切对应关系。这些基于功能的检测相对简单, 可以很容易地扩展到基于发现的高通量屏幕的真核细胞功能的调制器。

评估病原体和宿主编码因素对细胞感染的影响 使用上述感染条件, 在与培养的巨噬细胞共同培养1.5 小时后, 将有0.15 的输入野生类型lm恢复 (图 1a)。在随后的1.5 小时共同孵化 (感染后 3小时, hpi) 中, 可行的lm的恢复增加了 4倍, 从 3 hpi 增加了4倍到 6 hpi, 存活可行的lm的恢复又增加了7.5倍。由于细胞不溶于细...

Watch this Scientific Journal Video about Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses at JoVE.com

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

我们描述了可用于分析主机编码因子活性的体外和体内感染检测。

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

细胞内复制的微生物病原体最终必须退出受感染的细胞。其中，与宿主病原体相互作用的其他方面相比，相对研究不足。在这里，我们描述了分析病原体排放的技术。我们展示如何相对简单的检测可用于评估排放效率或描述后排放病原体。与建立任何实验系统一样，必须建立明确的控制措施，以确保在实验样本或队列中观察到真正的生物过程。演示这个程序将是彼得里亚盖尔，一个研究生从我的实验室。首先进行感染实验，使用移液器将20微升的新鲜准备的Lm inoculum添加到井中，盘中稍微倾斜，移液器尖端小心地放在上部介质中。避免使用移液器尖端接触井壁。轻轻移液每一个井的内容，以确保完全混合。请勿摇晃或明显倾斜板，以免将 LM 铺在井壁上。将细胞培养皿返回37摄氏度，5%的二氧化碳孵化器。使用未感染细胞的有条件介质作为稀释剂，每毫升基因霉素溶液中可准备 10 微克。感染后30分钟，小心地将30微升的根霉素溶液加入盘中，达到每毫升2点5微克的最终浓度。轻轻移液井的内容，混合并返回细胞培养皿到孵化器。要收获，请稍微倾斜盘子，并小心地使用移液器将整个介质从井中拆下。向井中加入五毫升的蒸馏无菌水。30秒后，将所得水酸盐转移到1点-5毫升微离心管和涡流中，用力进行10秒。将四点五微升和50微升水酸盐加入450微升蒸馏无菌水，以准备十倍和一百倍稀释。短暂涡流，确保彻底混合。将原10倍稀释和百倍稀释样品的50微升加入利原汤加糖板，然后使用板材摊铺器进行扩散。为了检测新兴的LM，从培养皿中提取10微升的覆盖介质，并分为两个五微升的等分和微型离心管。将 5 微升 DMEM/FCS 添加到一个等分中，并将含有每毫升 5 微升的 DMEM/FCS 的 5 微升添加到第二个等分项中作为控制。在室温下等待五分钟。在每个等分中加入90微升蒸馏无菌水，并大力旋转两个，10秒钟。然后在 LB 阿加板上添加 50 微升样品，然后使用板扩散器进行扩散。将盘子存放在37摄氏度的培养箱中两天，然后列举Lm菌落。使用自动细胞计数器，将准备好的二六四点七细胞的浓度调整为每毫升六细胞的浓度为 10 倍。将一毫升样品加入六孔组织培养板的井中。感染细胞与准备的Lm inoculum，对应于50的多重感染。感染后一点五小时，在充满500微升4%PFA的1点-5毫升微离心管中，从第一组油井收集介质，将管子放在冰上。要收集细胞内 Lm，请向每井加入一毫升蒸馏无菌水。60 秒后，将产生的水酸盐转移到 1 点 5 毫升微离心管中，并配有 500 微升的 4% PFA。用力旋转管子10秒钟，然后放在冰上。对于剩余的水井，去除介质，用每毫升5微克的DMEM/FCS代替，以杀死细胞外Lm.迅速将菜回孵化器。30 分钟后，取出介质，用 DMEM/FCS 更换，无需根霉素。再过两四个小时，收集第二次和第三次点到管中的介质，并把它们放在冰上冷却。10，000次G的离心管，7分钟。使用移液器，去除上流剂，而不干扰颗粒。将含有 50 微升 4% PFA 和 15 微升的 phaloidin 的染色鸡尾酒加入管中，混合以悬浮每个颗粒。在黑暗中孵育管，在4摄氏度下孵育20分钟。孵育后，将一毫升的FACS缓冲液添加到每个管中，并上下移液器混合。在离心机中旋转管，以10，000次G速度旋转7分钟。在不干扰颗粒的情况下去除上一液。立即使用，将每个颗粒悬浮在 400 微升 FAX 缓冲液中。如果存储供以后分析，请将 200 微升的 FAX 缓冲液和 4% PFA 的 200 微升添加到管中并混合以挂起。利用抗生素根霉素的短暂治疗中的感染条件，消除非内化Lm，在与培养的巨噬细胞共育1点5小时后，可恢复野生型Lm的零点-一至5%。在随后的联合孵化中，活体Lm的恢复量增加了四倍和七点五倍，这完全代表细胞内增殖。在初始感染期间，在三到六个 HPI 之间，观察到具有超维性prfA的Lm菌株的可比配置文件。当 prfA 从 Lm 菌株中删除时，在长时间的共育后，该菌株的恢复会减少。当六个HPI从井中去除根霉素时，一小时后可以检测到野生型和超维性prfA菌株的细胞外Lm，但无法检测出删除prfA菌株的菌株。覆盖未感染和受感染巨噬细胞的介质含有光散射、细菌大小的颗粒物。然而，只有来自受感染巨噬细胞的介质在尺寸门控内具有GP阳性信号。观察到感染的巨噬细胞出现法罗酮阳性的 Lm 的时间依赖性增加。当电镀48孔组织培养皿中的巨噬细胞时，留下一些没有细胞的井。然后，这些无细胞控制井在添加病原体、抗生素等方面与含细胞井完全相同，以确保实验信号依赖于宿主细胞的存在。与图一和图三所示的实验类似，可以测试病原体或宿主因子和或小分子对病原体细胞外流的影响。

Research

JoVE Journal

Immunology and Infection

Invasion of Human Cells by a Bacterial Pathogen

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 ...

科研

免疫与感染

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

“<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号：研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

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

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

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 ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...