Name: fluorescence microscopy methods for determining the viability of bacteria in association with mammalian cells
Uploaded: 2013-09-05T12:00:00
Description: Watch this Scientific Journal Video about Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells at JoVE.com

我们感谢阿霞斯米尔诺夫和Laura Gonyar的手稿的批判性阅读。这项工作是由美国国立卫生研究院资助R00 TW008042和R01 AI097312到AKCMBJ部分由美国国立卫生研究院的T32 AI007046被支承。

1。评估细菌存活与碘化丙啶和SYTO9 <ol><li>感染的细胞，粘附到12毫米直径的圆形盖玻片在24孔板中与感兴趣的细菌。 不修复的细胞与醛或有机溶剂。 </li><li>漂洗细胞一次轻轻地在0.1M 3 - （N-吗啉代）丙磺酸（MOPS），pH值7.2，含1mM的MgCl 2（MOPS / MgCl 2的）。 </li><li>孵育细胞在黑暗中10分钟，在室温下用的Alexa Fluor 647 -偶联抗体或凝集素，其结合到感兴趣的细菌菌种来说?...

<ol>
	<li>Woolard, M. D., Frelinger, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outsmarting+the+host:+bacteria+modulating+the+immune+response.">Outsmarting the host: bacteria modulating the immune response.</a> Immunol. Res. 41, 188-202 (2008).</li><li>Johnson, M. B., Criss, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+of+Neisseria+gonorrhoeae+to+neutrophils.">Resistance of Neisseria gonorrhoeae to neutrophils.</a> Front Microbiol. 2, 77 (2011).</li><li>Simons, M. P., Nauseef, W. M., Apicella, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Neisseria+gonorrhoeae+with+adherent+polymorphonuclear+leukocytes.">Interactions of Neisseria gonorrhoeae with adherent polymorphonuclear leukocytes.</a> Infect. Immun. 73, 1971-1977 (2005).</li><li>Seib, K. 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=Investigation+of+oxidative+stress+defenses+of+Neisseria+gonorrhoeae+by+using+a+human+polymorphonuclear+leukocyte+survival+assay.">Investigation of oxidative stress defenses of Neisseria gonorrhoeae by using a human polymorphonuclear leukocyte survival assay.</a> Infect. Immun. 73, 5269-5272 (2005).</li><li>Criss, A. K., Katz, B. Z., Seifert, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+of+Neisseria+gonorrhoeae+to+non-oxidative+killing+by+adherent+human+polymorphonuclear+leucocytes.">Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes.</a> Cell Microbiol. 11, 1074-1087 (2009).</li><li>Edwards, A. M., Massey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+cells+by+a+bacterial+pathogen.">Invasion of human cells by a bacterial pathogen.</a> J. Vis. Exp. (49), e2693 (2011).</li><li>Bozzola, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+specimen+preparation+techniques+for+transmission+electron+microscopy+of+cultured+cells.">Conventional specimen preparation techniques for transmission electron microscopy of cultured cells.</a> Methods Mol. Biol. 369, 1-18 (2007).</li><li>Dorward, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+bacteria-host+cell+interactions.">Ultrastructural analysis of bacteria-host cell interactions.</a> Methods Mol. Biol. 431, 173-187 (2008).</li><li>Yang, 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=Rapid,+absolute,+and+simultaneous+quantification+of+specific+pathogenic+strain+and+total+bacterial+cells+using+an+ultrasensitive+dual-color+flow+cytometer.">Rapid, absolute, and simultaneous quantification of specific pathogenic strain and total bacterial cells using an ultrasensitive dual-color flow cytometer.</a> Anal. Chem. 82, 1109-1116 (2010).</li><li>Mueller, R. S., 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=Vibrio+cholerae+strains+possess+multiple+strategies+for+abiotic+and+biotic+surface+colonization.">Vibrio cholerae strains possess multiple strategies for abiotic and biotic surface colonization.</a> J. Bacteriol. 189, 5348-5360 (2007).</li><li>Shen, 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=Enhanced+inactivation+of+Salmonella+and+Pseudomonas+biofilms+on+stainless+steel+by+use+of+T-128,+a+fresh-produce+washing+aid,+in+chlorinated+wash+solutions.">Enhanced inactivation of Salmonella and Pseudomonas biofilms on stainless steel by use of T-128, a fresh-produce washing aid, in chlorinated wash solutions.</a> Appl. Environ. Microbiol. 78, 6789-6798 (2012).</li><li>Hoefel, D., Grooby, W. L., Monis, P. T., Andrews, S., Saint, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+water-borne+bacteria+using+viability+assays+and+flow+cytometry:+a+comparison+to+culture-based+techniques.">Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques.</a> J Microbiol Methods. 55, 585-597 (2003).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+isolation+protocol.">Neutrophil isolation protocol.</a> J. Vis. Exp.. (17), e745 (2008).</li><li>Allen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+and+confocal+microscopy+of+neutrophils.">Immunofluorescence and confocal microscopy of neutrophils.</a> Methods Mol Biol. 412, 273-287 (2007).</li><li>Kaplan, E. L., Chhatwal, G. S., Rohde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+ability+of+penicillin+to+eradicate+ingested+group+A+streptococci+from+epithelial+cells:+clinical+and+pathogenetic+implications.">Reduced ability of penicillin to eradicate ingested group A streptococci from epithelial cells: clinical and pathogenetic implications.</a> Clin. Infect. Dis. 43, 1398-1406 (2006).</li><li>Chow, O. 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=Statins+enhance+formation+of+phagocyte+extracellular+traps.">Statins enhance formation of phagocyte extracellular traps.</a> Cell Host Microbe. 8, 445-454 (2010).</li><li>Thulin, P., 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=Viable+group+A+streptococci+in+macrophages+during+acute+soft+tissue+infection.">Viable group A streptococci in macrophages during acute soft tissue infection.</a> PLoS Med. 3, e53 (2006).</li><li>Kubica, 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=A+potential+new+pathway+for+Staphylococcus+aureus+dissemination:+the+silent+survival+of+S.+aureus+phagocytosed+by+human+monocyte-derived+macrophages.">A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages.</a> PLoS One. 3, (2008).</li><li>Swords, W. E., 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=Mycobacterium+xenopi+multiplies+within+human+macrophages+and+enhances+HIV+replication+in+vitro.">Mycobacterium xenopi multiplies within human macrophages and enhances HIV replication in vitro.</a> Microb. Pathog. 40, 41-47 (2006).</li><li>Tamilselvam, B., Almeida, R. A., Dunlap, J. R., Oliver, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+uberis+internalizes+and+persists+in+bovine+mammary+epithelial+cells.">Streptococcus uberis internalizes and persists in bovine mammary epithelial cells.</a> Microb. Pathog. 40, 279-285 (2006).</li><li>Martinez, A. 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=Molecular+determination+of+Mycobacterium+leprae+viability+by+use+of+real-time+PCR.">Molecular determination of Mycobacterium leprae viability by use of real-time PCR.</a> J. Clin. Microbiol. 47, 2124-2130 (2009).</li><li>Botha, M., Botes, M., Loos, B., Smith, C., Dicks, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactobacillus+equigenerosi+strain+Le1+invades+equine+epithelial+cells.">Lactobacillus equigenerosi strain Le1 invades equine epithelial cells.</a> Appl. Environ. Microbiol. 78, 4248-4255 (2012).</li><li>Allen, L. A., Schlesinger, L. S., Kang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulent+strains+of+Helicobacter+pylori+demonstrate+delayed+phagocytosis+and+stimulate+homotypic+phagosome+fusion+in+macrophages.">Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages.</a> J. Exp. Med. 191, 115-128 (2000).</li><li>Smith, C. D., Berk, S. G., Brandl, M. T., Riley, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+characteristics+of+diarrheagenic+Escherichia+coli+pathotypes+and+Helicobacter+pylori+during+passage+through+the+free-living+ciliate,+Tetrahymena+sp.">Survival characteristics of diarrheagenic Escherichia coli pathotypes and Helicobacter pylori during passage through the free-living ciliate, Tetrahymena sp.</a> FEMS Microbiol. Ecol. 82, 574-583 (2012).</li><li>Morse, S. A., Bartenstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purine+metabolism+in+Neisseria+gonorrhoeae:+the+requirement+for+hypoxanthine.">Purine metabolism in Neisseria gonorrhoeae: the requirement for hypoxanthine.</a> Can. J. Microbiol. 26, 13-20 (1980).</li><li>Johnson, M. B., Criss, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+gonorrhoeae+phagosomes+delay+fusion+with+primary+granules+to+enhance+bacterial+survival+inside+human+neutrophils.">Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils.</a> Cell Microbiol. , (2013).</li><li>Stocks, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+use+of+the+commercially+available+viability+stain.">Mechanism and use of the commercially available viability stain.</a> BacLight. Cytometry A. 61, 189-195 (2004).</li></ol>

这里呈现的是使用DNA结合和可行性染料与荧光凝集素识​​别连接到内部和人体细胞活的和死的细菌结合两种协议。由于这两种协议都有效地死菌歧视的活，要使用哪个协议的选择取决于实验的目的。第一个协议使用碘化丙啶检测不能存活的细菌和SYTO9检测的细菌。 如图4A所示是方案1的代表性图像施加到感染N.人中性粒细胞淋球菌 。这些染料结合的凝集素，大豆凝集素?...

有一个动态的互动和在其居住的细菌和宿主之间的共同进化。细菌已经进化粘附细胞器，分泌系统，和/或产生毒素，使宿主吞噬细胞和非吞噬细胞的生产性感染的能力。此外，细菌还必须与认可与宿主免疫系统的抗菌活性。宿主的免疫系统包括先天性和适应性组件，包括物理和化学屏障，免疫细胞，补体系统，免疫和体液免疫的其它部件。虽然许多细菌很容易受到杀伤和清除由多层宿主的免疫反应，一些致病性和投机细菌已经进化机制来感染多种宿主细胞和宿主的免疫反应1颠覆间隙。 淋球菌是一种细菌病原体的一个例子这是高度适应坚持其人类宿主。Ñ。淋病容易定殖泌尿生殖道癌，咽癌，结膜，和直肠的粘膜上皮细胞的腔表面。定植触发嗜中性粒细胞的大量招聘在粘膜部位。中性粒细胞是专业的吞噬细胞是具有多种抗微生物过程，以杀死微生物，但是，N。淋病是能够存活在嗜中性粒细胞2-5的存在。了解如何细菌病原体如N。淋球菌颠覆，压制，并劫持了免疫反应通常在恶劣的环境中主机的最终生存是至关重要的防治传染性疾病的新疗法的发展。 常用于宿主细胞研究细菌生存实验的协议包括菌落总数测定，庆大霉素保护法和电子显微镜。在菌落计数测定法，受感染的细胞群被裂解（例如，用洗涤剂的whICH细菌有抗药性）解放细菌。将裂解物稀释并涂布在琼脂上的媒体，以及集落形成单位中的溶胞产物被列举为每个时间点和/或实验条件。此方法报道的全部细菌群体的生存力，但并不能够胞内和胞外生存区分的。上的菌落计数测定法，庆大霉素保护测定法，一种变化明确地测量细胞内细菌存活，基于抗生素庆大霉素无法越过的真核细胞膜6。但是，这种测定法是依赖于细菌会受影响杀庆大霉素（或另一种抗生素是类似的真核细胞膜透性）和抗生素的无力获得内部的细菌。因此，庆大霉素保护法可能无法有效检查所有细菌种类或检查高度细菌生存时胞饮细胞如嗜中性粒细胞。这些方法都不揭示了亚细胞定位或个人的细菌等行为（ 例如 ，如果细菌形成聚集体或行为不同于单个细菌细胞菌落）。另一种经常使用的方法，检查单个外部和内部的细菌的生存能力是薄截面的透射电子显微镜（TEM）。这种方法的优点在于，它可以提供有关该细菌在宿主细胞中的位置（ 例如，吞噬体，细胞质，自噬体），它可以被进一步研究通过免疫电镜用金偶联抗体亚细胞标记信息。然而，电子显微镜是不是在评估细菌生存力特别敏感。当包埋切片进行染色用醋酸双氧铀，柠檬酸铅，或其他电子密度试剂和通过电子显微镜成像，电子致密的细菌被认为是可行的和电子角TRON朗讯不能存活7,8。然而，这个假设高估细菌生存能力，因为只有那些死去的细菌严重破坏细胞膜和细胞质泯灭的出现电子透亮。此外，某些细菌种可能显示的范围内的电子密度取决于生长的阶段，因此很难确定可行性。 作为替代或除了这些广泛使用的方法，在这里我们提供的协议和原理为利用荧光染料，表明细菌生存力评估附着并通过宿主细胞内在化细菌的存活。以识别细胞外的细菌，感染的细胞首先暴露于荧光试剂，如凝集素或菌特异性抗体。然后将感染的细胞透化，并暴露于DNA特异性染料是差分访问的细菌与完整与退化的膜，作为替代的细菌存活率。在第一protocol，膜渗透性染料SYTO9识别细菌总人口，而碘化丙啶是只对那些破坏细胞膜，因此被认为无法存活的细菌。碘化丙锭和SYTO9已被用于评价细菌存活在生物膜中，判别从非致病性细菌致病，并且列举可行的水性细菌9-12。在第二协议，4&#39;，6&#39;-二脒基-2 - 苯基吲哚（DAPI）确定总的细菌，而SYTOX Green是唯一可访问的无活力的人口。这些活力染料对可与免疫有关的感兴趣的蛋白质，以确定各细菌的位置，例如以限定细菌亚细胞定位进行组合。利用这些分析提供关键洞察导致宿主细胞的感染过程中杀死细菌或生存的相互作用。在这篇文章中列出的协议被用来评估的可行性是连接到与内初级人嗜中性粒细胞，包括中性粒细胞的吞噬体5,13,14的不同人群淋病奈瑟氏球菌 。然而，这些协议可以被用于评估在专业的吞噬细胞，非专业吞噬细胞和原生动物15-24的革兰氏阳性和革兰氏阴性细菌的生存能力。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>21 G 3/4 butterfly needles for blood collection</td>
			<td>Becton Dickinson</td>
			<td>367251</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes with Sodium Heparin 10 ml</td>
			<td>Becton Dickinson</td>
			<td>366480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water for irrigation</td>
			<td>Baxter</td>
			<td>07-09-64-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran 500</td>
			<td>Sigma</td>
			<td>31392</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S641</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Ricca Chemical Company</td>
			<td>RDCD0200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS no Ca2+ or Mg2+</td>
			<td>Thermo Scientific</td>
			<td>SH3002802</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll solution</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher Scientific</td>
			<td>BP2401</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm circular glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-80 12CIR-1</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Corning Incorporated</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>Pooled Human Serum</td>
			<td>Sigma</td>
			<td>S7023</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Mediatech</td>
			<td>15-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr>
			<td>Human interleukin-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Scientific</td>
			<td>BP214</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Life Technologies</td>
			<td>L7007 or L7012</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTO9</td>
			<td>Life Technologies</td>
			<td>L7007 or L7012</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Fluka Analytical</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-coupled soybean lectin</td>
			<td>Life Technologies</td>
			<td>L-32463</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Green</td>
			<td>Life Technologies</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-CD63</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>H5C6</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Antibody Labeling Kit</td>
			<td>Life Technologies</td>
			<td>A20187</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer Bright Line</td>
			<td>Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>EMS</td>
			<td>78320</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend RT + Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004377</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence microscopy methods for determining the viability of bacteria in association with mammalian cells

中央细菌发病机理的字段是定义是否以及如何暴露于真核细胞中后微生物生存的能力。目前的协议来解决这些问题包括菌落总数测定，庆大霉素保护法和电子显微镜。菌落总数和庆大霉素保护法只评估整个细菌群体的生存能力，也就无法确定单个细菌的生存能力。电子显微镜可以用于确定个体的细菌的生存能力和提供关于其在宿主细胞中的定位信息。然而，细菌常常显示一个范围的电子密度，使得可行性评估困难。本文概述了使用，揭示内部以及与宿主细胞相关的各个细菌的生存能力荧光染料的协议。这些分析是独自开发，以评估在小学人类嗜中性粒细胞淋球菌存活，但应该是一个pplicable任何细菌宿主细胞的相互作用。这些协议结合膜可渗透荧光染料（SYTO9和4&#39;，6 - 二脒基-2 - 苯基吲哚[DAPI]），其染色所有的细菌，具有不可透过膜的荧光染料（碘化丙锭和SYTOX绿），这是唯一的访问不能存活的细菌。前的真核细胞透化，抗体或荧光试剂加入到识别细胞外细菌。因而这些测定鉴别细菌粘附于和里面的真核细胞的生存能力。一种协议还提供了使用的可行性染料结合的荧光抗体的真核细胞的标记物，以确定个别细菌的亚细胞定位。这篇文章中讨论的细菌存活染料是一个敏感的补充和/或替代传统的微生物学技术来评估个体细菌的生存能力，并提供有关在细菌的宿主细胞生存信息秒。

概括的协议被用来研究N的生存淋球菌暴露于人原后中性粒细胞5,26。中性粒细胞被感染N.淋病和与方案1进行处理，利用绿色荧光活力染料SYTO9和红色荧光碘化丙啶（ 图4A）。染料被加入以皂角苷的存在下，该螯合胆固醇优先透化的宿主细胞的质膜，而不是全淋球菌膜。其他洗涤​​剂可能需要进行测试，如果感兴趣的细菌的细胞膜含有高量的胆固醇?...

Watch this Scientific Journal Video about Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells at JoVE.com

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

中央细菌发病机理的字段是定义是否以及如何暴露于真核细胞中后微生物生存的能力。本文概述了使用，揭示内部以及与宿主细胞相关的各个细菌的生存能力荧光染料的协议。

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

Research

JoVE Journal

Immunology and Infection

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 ...

科研

免疫与感染

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

听话的哺乳动物细胞原生动物引发的细菌感染

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

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. ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...