Name: immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells
Uploaded: 2017-07-03T13:00:00
Description: Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

PS是比尔和梅琳达·盖茨大挑战奖牌OPP1141322的收件人。 PV由瑞士国家科学基金会早期博士后移动奖学金和Roux-Cantarini博士后研究金支持。 PJS由HHMI授权和ERC-2013-ADG 339579解密支持。

1.细菌和宿主细胞的制备<ol><li>使用无菌镊子分别在24孔或12孔板中转移12或14毫米玻璃盖，每个实验条件计数一个孔。在组织培养罩中通过UV处理灭菌15分钟。 注意：包括用于细菌攻击的控制井和通过外源胁迫进行SG诱导。 </li><li>对于具有12mm盖玻片的24孔板，将1×10 5个哺乳动物细胞（ 例如 HeLa细胞）种在盖玻片上;用无菌吸头向下压盖玻片。让细胞在37℃下在适合每种细胞类型?...

<ol>
	<li>Reineke, L. C., Lloyd, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversion+of+stress+granules+and+P-bodies+during+viral+infection.">Diversion of stress granules and P-bodies during viral infection.</a> Virology. 436 (2), 255-267 (2013).</li><li>Kedersha, N., Ivanov, P., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules+and+cell+signaling:+more+than+just+a+passing+phase.">Stress granules and cell signaling: more than just a passing phase.</a> Trends Biochem Sci. 38 (10), 494-506 (2013).</li><li>Vonaesch, P., Campbell-Valois, F. -. X., Dufour, A., Sansonetti, P. J., Schnupf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+flexneri+modulates+stress+granule+composition+and+inhibits+stress+granule+aggregation.">Shigella flexneri modulates stress granule composition and inhibits stress granule aggregation.</a> Cell Microbiol. 18 (7), 982-997 (2016).</li><li>Kolobova, E., Efimov, A., 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=Microtubule-dependent+association+of+AKAP350A+and+CCAR1+with+RNA+stress+granules.">Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules.</a> Exp Cell Res. 315 (3), 542-555 (2009).</li><li>Anderson, P., Kedersha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules:+the+Tao+of+RNA+triage.">Stress granules: the Tao of RNA triage.</a> Trends in biochemical sciences. 33 (3), 141-150 (2008).</li><li>Anderson, P., Kedersha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressful+initiations.">Stressful initiations.</a> J Cell Sci. 115, 3227-3234 (2002).</li><li>Mohr, I., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+translation+at+the+nexus+of+infection+and+immunity.">Host translation at the nexus of infection and immunity.</a> Cell Host Microbe. 12 (4), 470-483 (2012).</li><li>Hu, S., Claud, E. C., Musch, M. W., Chang, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granule+formation+mediates+the+inhibition+of+colonic+Hsp70+translation+by+interferon-+and+tumor+necrosis+factor.">Stress granule formation mediates the inhibition of colonic Hsp70 translation by interferon- and tumor necrosis factor.</a> Am J Physiol Gastointest Liver Physiol. 298 (4), 481-492 (2010).</li><li>Barry, E. M., Pasetti, M. F., Sztein, M. B., Fasano, A., Kotloff, K. L., Levine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+pitfalls+in+Shigella+vaccine+research.">Progress and pitfalls in Shigella vaccine research.</a> Nat Rev Gastroenterol Hepatol. 10 (4), 245-255 (2013).</li><li>Lanata, C. F., Fischer-Walker, C. L., 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=Global+Causes+of+Diarrheal+Disease+Mortality+in+Children.">Global Causes of Diarrheal Disease Mortality in Children.</a> PloS One. 8 (9), 72788 (2013).</li><li>Ashida, H., Ogawa, 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=Shigella+deploy+multiple+countermeasures+against+host+innate+immune+responses.">Shigella deploy multiple countermeasures against host innate immune responses.</a> Curr Opin Microbiol. 14 (1), 16-23 (2011).</li><li>Ashida, H., Ogawa, M., Mimuro, H., Kobayashi, T., Sanada, T., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+are+versatile+mucosal+pathogens+that+circumvent+the+host+innate+immune+system.">Shigella are versatile mucosal pathogens that circumvent the host innate immune system.</a> Curr Opin Immunol. 23 (4), 448-455 (2011).</li><li>Kedersha, N., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+stress+granules+and+processing+bodies.">Mammalian stress granules and processing bodies.</a> Methods Enzymol. 431, 61-81 (2007).</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> Nat Re. Micro. 7 (5), 333-340 (2009).</li></ol>

本文概述的方案描述了在存在或不存在外源胁迫的情况下，非感染细胞和感染细胞溶质病原体柔毛霉的细胞中的SG的诱导，定位和分析。使用免费的成像软件，协议允许对SG形成进行精确的定性和定量分析，以鉴别和统计学地解决给定表型的差异。 在感染，SG感应和成像部件的协议中有几个关键步骤。对于感染，重要的是与宿主细胞的细菌相互作用，如本文中概述的方?...

在细胞水平上可视化宿主 - 病原体相互作用是获得对致病性策略和识别关键细胞途径的见解的有力方法。事实上，病原体可以用作确定重要细胞靶点或结构的工具，因为病原体已经发展为颠覆中枢细胞过程，作为其自身存活或繁殖的策略。可以通过重组表达荧光标记的宿主蛋白来实现细胞成分的可视化。虽然这允许进行实时分析，但具有特异标记宿主蛋白质的细胞系的产生是非常费力的并且可能导致不期望的副作用。使用特异性抗体检测细胞因子更方便，因为可以同时分析多个宿主因子，并且不限于特定的细胞类型。缺点是只有静态视图可以被捕获，因为免疫荧光分析需要宿主细胞修复离子。然而，免疫荧光成像的一个重要优点是其易于定性和定量分析。这又可用于获得统计学显着的差异，以提供对宿主 - 病原体相互作用的新见解。 荧光图像分析程序是执行3D和4D分析的强大的分析工具。然而，软件及其维护成本高昂使得基于免费开源软件的方法更具吸引力。使用生物分析软件的仔细图像分析是有价值的，因为它证实了视觉分析，并且在分配统计学意义时，增加了给定表型的正确性的置信度。以前，使用免费的ImageJ软件分析SG，这需要手动识别个人SG 4 。在这里，我们提供了一个在细菌SG形成诱导和分析方案使用免费的开源生物图像分析软件ICY（http://icy.bioimageanalysis.org）进行真实感染。生物图像分析软件具有高度适用于SG分析的内置点检测程序。它允许在指定的感兴趣区域（ROI）中对自动检测过程进行微调。这克服了对个别SG的手动分析的需要，并消除了抽样偏差。 许多环境胁迫引起SGs的形成，SG是直径为5 - 6μm的相间致密的胞质结构，非膜结构为0.2-5μm。这种细胞反应在酵母，植物和哺乳动物中进化上保守，并且当全球蛋白质翻译被抑制时发生。它涉及停滞的翻译起始复合物聚集到SG中，其被认为是用于翻译失活的mRNA的保留位置，允许细胞mRNA的子集的选择性翻译。消除压力后，SGs溶解，蛋白质合成的全球速率恢复。 SG由翻译延伸起始因子，涉及RNA代谢的蛋白质，RNA结合蛋白质以及支架蛋白质和参与宿主细胞信号传导的因子2组成 ，尽管精确的组成可以根据施加的应力而变化。诱导SG形成的环境因素包括氨基酸饥饿，紫外线照射，热休克，渗透压休克，内质网应激，缺氧和病毒感染2,7,8 。在理解病毒如何诱导和颠覆SG形成方面取得了许多进展，而关于其他病原体（如细菌，真菌或原生动物病原体）如何影响细胞应激反应1,7尚不清楚。 师哥lla flexneri是人类的革兰氏阴性兼性细胞溶质病原体和严重腹泻或志贺氏菌病的致病因子。志贺氏菌病是一个主要的公共卫生负担，每年导致5岁至9岁的儿童死亡28 000人。 S. flexneri感染结肠上皮，并通过劫持宿主的细胞骨架成分11,12扩散细胞对细胞。上皮细胞的感染支持了胞内溶胶酶的复制，但被感染的巨噬细胞通过炎性细胞死亡过程称为焦灼死亡。感染导致大量招募中性粒细胞和严重的炎症，伴随着热，氧化应激和组织破坏。因此，虽然被感染的细胞受到感染引起的内部应激，例如高尔基体破坏，遗传毒性应激和细胞骨架重排感染细胞也由于炎症过程而受到环境压力。 使用许多SG标记物表征S. flexneri感染对细胞对环境胁迫反应能力的影响已经证明，感染导致SG组成3的定性和定量差异。然而，其他细菌病原体知之甚少。在这里，我们描述了一种感染宿主细胞与细胞溶质病原体S. flexneri ，不同环境胁迫的细胞的应激，SG组分的标记，以及感染SG的形成和组成的定性和定量分析的方法和非感染细胞。该方法广泛适用于其他细菌病原体。此外，SG形成的图像分析可以用于病毒或其他病原体的感染。可用于分析SG感染后的形成或感染对SG形成的影响以响应外源性应激。

<table border="0" cellpadding="0" cellspacing="0" width="1191">
	<colgroup>
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Primary Antibodies</td>
			<td style="width:211px;"></td>
			<td style="width:76px;"></td>
			<td style="width:672px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3b (N20), origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-16377</td>
			<td>Robust and widely used SG marker. Cytosolic staining allows cell delineation. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3b (A20), origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-16378</td>
			<td>Same target as eIF3b (N20) and in our hands was identical to eIF3b (N20). Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3A (D51F4), origin rabbit (MC: monoclonal)</td>
			<td>Cell Signaling</td>
			<td>3411</td>
			<td>Part of multiprotein eIF3 complex with eIF3b . Dilution 1 in 800</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4AI, origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-14211</td>
			<td>Recommended by (Ref # 13). Dilution 1 in 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4B, origin rabbit</td>
			<td>Abcam</td>
			<td>ab186856</td>
			<td>Good stress granule marker in our hands. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4B, origin rabbit</td>
			<td>Cell Signaling</td>
			<td>3592</td>
			<td>Recommended by Ref # 13. Dilution 1 in 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4G, origin rabbit</td>
			<td>Santa Cruz</td>
			<td>sc-11373</td>
			<td>Widely used SG marker. (Ref # 13): may not work well in mouse cell lines. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G3BP1, origin rabbit (MC: monoclonal)</td>
			<td>BD Biosciences</td>
			<td>611126</td>
			<td>Widely used SG marker. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tia-1, origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-1751</td>
			<td>Widely used SG marker. Can also be found in P bodies when SG are present (Ref # 13). Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Alexa-conjugated Secondary Antibodies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-goat , origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-11055</td>
			<td>Cross absorbed. Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-goat, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-11057</td>
			<td>Cross absorbed. Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-21202</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A10037</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A647 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A31571</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-rabbit, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-21206</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-rabbit, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A10042</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shigella flexneri</td>
			<td></td>
			<td></td>
			<td>Available from various laboratories by request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone Casein Soya (TCS) broth</td>
			<td>BD Biosciences</td>
			<td>211825</td>
			<td>Standard growth medium for Shigella, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCS agar</td>
			<td>BD Biosciences</td>
			<td>236950</td>
			<td>Standard growth agar for Shigella, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Congo red</td>
			<td>SERVA Electrophoresis GmbH</td>
			<td>27215.01</td>
			<td>Distrimination tool for Shigell that have lost the virulence plasmid, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly L lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1274</td>
			<td>Useful to coating bacteria to increase infection, application - infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>Selective killing of extracellular but not cytosolic bacteria, application - infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Life Technologies</td>
			<td>15630-056</td>
			<td>PH buffer useful when cells are incubated at room-temperature, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>31885</td>
			<td>Standard culture medium for HeLa cells, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Biowest</td>
			<td>S1810-100</td>
			<td>5% supplementation used for HeLa cell culture medium, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td>11140</td>
			<td>1/100 dilution used for HeLa cell culture medium, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Reagent diluent, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium arsenite</td>
			<td>Sigma-Aldrich</td>
			<td>S7400</td>
			<td>Potent stress granule inducer (Note: highly toxic, special handling and disposal required), application - stress inducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clotrimazole</td>
			<td>Sigma-Aldrich</td>
			<td>C6019</td>
			<td>Potent stress granule inducer (Note:health hazard, special handling and disposal required), application - stress inducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Electron Microscopy Scences</td>
			<td>15714</td>
			<td>4% PFA is used for standard fixation of cells, application - fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used at 0.03% for permeabilizationof host cells before immunofluorescent staining, application - permeabilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A647-phalloidin</td>
			<td>Thermo Fisher</td>
			<td>A22287</td>
			<td>Dilution is at 1/40, best added during 2ary antibody staining, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Nucleid acid stain used to visualize both the host nucleus and bacteria, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>BR701501</td>
			<td>Paraffin film useful for immunofluorescent staining of coverslips, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold</td>
			<td>Thermo Fisher</td>
			<td>36930</td>
			<td>Robust mounting medium that works well for most fluorophores , application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mowiol</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>Cheap and robust mounting medium that works well for most fluorophores, application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well cell culture plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td>Standard tissue culture plates, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-mm glass coverslips</td>
			<td>NeuVitro</td>
			<td>1001/12</td>
			<td>Cell culture support for immunofluorescent applications, application - cell support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forceps</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>Cheap and obust mounting medium that works well for most fluorophores, application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Programs and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Data analysisand graphing program with robust statistical test options, application - data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Confocal microsope, application - image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>Professional image analysis program, application - data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Data analysis and graphing program, application - data analysis</td>
		</tr>
	</tbody>
</table>

immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells

细胞成分的荧光成像是调查宿主 - 病原体相互作用的有效工具。病原体可以影响感染细胞的许多不同特征，包括细胞器超微结构，细胞骨架网络组织，以及细胞过程如应激颗粒（SG）形成。病原体如何颠覆宿主过程的特征是发病机理领域的重要和组成部分。虽然可变表型可能容易可见，但是由病原体攻击诱导的细胞结构中的定性和定量差异的精确分析对于定义实验和对照样品之间的统计学显着性差异至关重要。 SG形成是进化上保守的应激反应，导致抗病毒反应，长期以来使用病毒感染进行了研究1 。 SG形成也影响信号级联，可能还有其他未知的结果适应2 。对病毒以外的病原体如细菌病原体的这种应激反应的表征目前是一个新兴的研究领域3 。目前，即使在病毒系统中，SG形成的定量和定性分析尚未被常规使用。在这里，我们描述了诱导和表征未感染细胞中SG形成和细胞感染细胞病原体的细胞的简单方法，其影响响应于各种外源胁迫的SG的形成。 SG形成和组成的分析通过使用许多不同的SG标记和ICY的点检测器插件（开源图像分析工具）来实现。

为了解释和说明本手册中描述的方案，我们将感染或不与细胞溶质病原体S. flexneri的 HeLa细胞中克霉唑诱导的SGs的形象进行了表征。程序概述如图1所示 ，包括在刚果红板上分解的毒力和无毒的柔毛菌 ，细菌制备，感染，环境应激的添加，样品固定和染色，样品成像和定量，以及图像分析。可以使用许多不同的应力来诱导SG形成，?...

Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

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

我们描述了在用细菌和许多不同的应力攻击细胞后，哺乳动物细胞中应激颗粒形成的定性和定量分析的方法。该方案可用于研究广泛的宿主细菌相互作用中的细胞应激颗粒反应。

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 ...

The overall goal of this experiment is to assess the effects of a bacterial infection on the ability of host cells to form stress granules to response to exogenous stress. This method is a valuable tool in the field of cellular microbiology for assessing whether or not a given pathogen is able to alter or subvert the whole stress granule pathway. The main advantage of this technique is that stress granules can be qualitatively and quantitatively be analyzed in a rigorous and automated manner using free image analysis programs.This methods provides insight into the dynamics of stress granule formation and composition and how these parameters are affected by a specific pathogen. Thank you. Before beginning the challenge, inoculate a red S.flexneri M90T bacteria colony from a freshly streaked tryptic soy agar 1%Congo red agar plate into a 15 milliliter conical tube, containing eight milliliters of tryptic soy broth.After tightening the lid, incubate the colony overnight with shaking at 222 RPM and 30 degrees Celsius. The next day, subculture 150 microliters of the bacteria in eight milliliters of fresh tryptic soy broth for about two hours at 37 degrees Celsius and 222 RPM to initiate a late exponential phase induction of virulence gene expression and to enhance the bacterial infectability. When the optical density of the subculture is between 0.6 and 0.9, transfer one milliliter of the bacteria into a 1.5 milliliter tube for their collection by centrifugation.And re-suspend the pellet in infection medium at an optical density of one. Next, aspirate the supernatants from each well of a HeLa cell coverslip culture. And carefully wash the HeLa cells with 500 microliters of fresh room temperature HeLa cell medium per well.Replace the wash with 500 microliters of infection medium per well, and centrifuge the 24 well plate to spin the bacteria onto the cells. Transfer the settled bacterial cocultures into a 37 degree Celsius tissue culture incubator for 30 minutes to facilitate the host cell infection. At the end of the incubation, remove the exogenous bacteria from the cells with three rinses in one milliliter of fresh 37 degree Celsius HeLa culture medium per wash.Then cover the infected cells with one milliliter of fresh culture medium containing gentamicin and return the plate to the cell culture incubator. At the end of the incubation, replace the medium with 500 microliters of the appropriate culture medium, supplemented with the stressor of interest, and return the cells to the tissue culture incubator. After one hour, aspirate the supernatant completely, and fix the samples in 0.5 milliliters of room temperature 4%paraformaldehyde in PBS for 30 minutes.Next, discard the fixative into the appropriate waste container, and wash the coverslips with one milliliter of tris-buffered saline for two minutes with gentle shaking. At the end of the wash, completely aspirate the TBS and permeabilize the cells in each well with 500 microliters of 0.3%Triton X-100 in TBS. After 10 minutes, replace the permeabilizing solution with one milliliter of TBS, swirl the plate gently, and replace the TBS with one milliliter of blocking solution for one hour at room temperature.To label the cells with a primary antibody cocktail of interest, spot 50 microliters of the primary antibody mix per coverslip onto a piece of plastic Parafilm firmly taped onto the bench top. Then use tweezers to pick up the first coverslip. And dab the edge of the coverslip onto a piece of tissue paper to remove the excess liquid.Carefully place the coverslip onto the drop and incubate the coverslips under the appropriate labeling conditions. At the end of the primary antibody labeling incubation, transfer each coverslip into an individual well of a new 24 well plate containing one milliliter of TBS per well, and wash the cells at room temperature on a plate shaker for five minutes three times. After the last wash, label the cells on each coverslip with the appropriate secondary antibody cocktail mix, as just demonstrated.And incubate the coverslips in the dark for one hour at room temperature. At the end of the secondary antibody labeling incubation, wash the cells in one milliliter of PBS three times in a new 24 well plate with gentle shaking as just demonstrated, but protected from light. After the last wash, briefly dip each coverslip in deionized water to remove the salt, dab the coverslip edges onto a tissue, and carefully mount the coverslips onto five microliters of fluorescence mounting medium per glass microscope slide without bubbles.Then seal the coverslip with nail polish. And store the slides as appropriate until imaging. To image the cells by fluorescence confocal microscopy, begin by selecting the appropriate objective and the appropriate fluorescent channels for image acquisition.Then acquire image stacks of the cells, using the appropriate imaging software. When all of the images have been captured, use the appropriate imaging analysis software to collapse the image stacks and save the stacks as TIFF files. Next, load a control and an experimental TIFF image to the image analysis platform, and select Lookup Table to open the channel parameters in the Sequence window.Click on all of the channel tab checkboxes to deactivate all of the channels. Then click channel zero, and click on the channel zero color bar to select the preferred color, increasing the intensity of the channel as necessary to visualize all of the structures. After selecting and adjusting each of the appropriate color channels for the experimental analysis, select the canvas tab and adjust the zoom tab to visualize about 10 cells per viewing field.Using the polygon tool, click on the cell of interest and extend the line around the cell to delineate the cell boundary. To name this region of interest, in the region of interest window, right-click on the cell of interest, and double-click on the region of interest's name to modify it. After selecting and naming all of the cells of interest in the same manner, open the Spot Detector application within the detection and tracking tab.Open the Pre Processing tab and select the channel to be analyzed. Within the Detector tab, select Detect bright spots over dark background and select the appropriate scale and sensitivity. Under the Region of Interest tab, select the suggested region of interest from the sequence.Under the Filtering tab, select NoFiltering. In the Output tab, select the appropriate output. Select Remove previous spots rendered as regions of interest and click no file selected to select the folder for saving the file.Then under the Display tab, select the appropriate options of interest and click Start Detection. Within the imaging analysis software, the spot detector can be used to identify the stress granules within each of the designated regions of interest. A binary image can then be generated to display all of the identified stress granules.Stress granule analysis must be carefully tailored to each stress granule market analyzed. For example, changing the size requirement of the region of interest detected, or changing the sensitivity of the detection parameters lead to varying results. At higher pixel size scales, smaller stress granules will not be counted, and the numbers of stress granules will decrease.While increasing the sensitivity results in an increase in the number of stress granules. For example, in this experiment, a scale of two with a sensitivity of 100 gave the best result from the stress granule marker G3BP1. While a scale of two with a sensitivity of 55 gave the best result for the EIF3B stress granule marker.The surface area can then be calculated from the size of the stress granules. Frequency distribution plots are useful for highlighting shifts in the size of stress granules between different cell populations. In addition, the intensity of the fluorescence can provide information about the quality of the stress granules analyzed.Once mastered, the whole cell challenge can be done in about six to seven hours, and the analysis can be completed in another two to three hours. While attempting this procedure, it's important to remember to always process the control and the experimental samples at the same time and under the same conditions to minimize variability within the data. Following this procedure, the analysis can also be extended to a three-dimensional volume to get additional insights into stress granule localization.This semi-automated procedure allows for a rigorous and unbiased analysis of stress granules in uninfected and infected cells. It can reveal previously unknown subversions of the stress granule pathway. After watching this video, you should have a good understanding of how to infect cells with bacteria, how to induce stress granule formation, and how to use a semi-automated approach for analyzing stress granules in a qualitative and quantitative manner.And don't forget that with Shigella flexneri and stress granule inducing agents like clotrimazole can be hazardous, and that precautions such as wearing gloves, working in a BL2 lab, and properly discarding all of the reagents are necessary.

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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 ...