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，在MOPS / MgCl 2的，以检测外部细菌。 </li></ol>注：行为与活的和死的细菌控制，在没有宿主细胞，表明感兴趣的凝集素或抗体结合所有的细菌，无论生存能力（ 图1）。 <ol start="4"><li>冲洗细胞两次，MOPS / 氯化镁 。 </li><li>吸米EDIA从细胞和加入0.5毫升活/死染色溶液。活/死染色溶液为5微米SYTO9，30微米碘化丙啶，并在MOPS / 氯化镁 0.1％皂素（终浓度）。 </li><li>孵育细胞15分钟，在室温下在黑暗中。 </li><li>冲洗细胞两次在公开资讯观测站/ 氯化镁。 </li><li>反转盖玻片面朝下放置玻片和密封用透明指甲油。不要使用安装媒体。 </li><li>在30分钟内采集图像，使用荧光显微镜与绿色，红色和远红色图像采集兼容的过滤器集。 </li></ol>注意：两个常规荧光显微镜和共焦激光扫描显微镜可以使用。经过30分钟的荧光染料开始从细菌到泄漏和获取的数据不再准确。本文中显示图像，在使用的OpenLAB软件滨松的Orca-ER数码相机尼康的Eclipse E800直立荧光显微镜收购。 650纳米和663的发射滤波器 - - 使用过滤器以590激发波长检测的Alexa Fluor 647的荧光735纳米，并且是伪色的蓝色。 580纳米和600的发射滤波器 - - 660纳米使用过滤器以540激发波长检测从碘化丙啶荧光。 495 nm和515发射滤光片 - - 555 nm处使用过滤器具有465激发波长检测从SYTO9荧光。 <ol start="10"><li>该协议将导致在外部不能存活的细菌出现蓝色+红色图像，内部自生能力的细菌仅出现红色，外​​部活菌出现蓝+绿，内部活菌只呈现绿色。由眼计数的细菌是没有自生能力的外部，内部没有自生能力，外部可行的，并且内部可行的数字。 </li><li>通过将外部的活菌数由外部细菌的总数（计算外部活菌百分比可行加nonvi能）。通过将内部的活菌数由内部的细菌总数（可行加上不能存活）（ 图4）计算出内部的存活细菌的百分比。 </li><li>有两个基本的控制与此协议来执行。首先，验证所有的自生能力的细菌是碘化丙啶阳性和所有活细菌是SYTO9阳性。细菌（在没有任何宿主细胞）在对数中期文化应&gt; 95％SYTO9阳性。 如图2中所示为N的对数中期培养淋病 （10 8菌落形成每毫升为单位）孵育活/死染色溶液。第二，验证这两个碘化丙锭和SYTO9可以进入通透，感染的宿主细胞。 <ol><li>为了产生死菌的群体，收集2×10 8菌落形成单位在1.5 ml离心管中，加入70％的异丙醇和坐10分钟。沉淀的细菌在微量一个第二次洗，细菌在PBS中以除去残留的异丙醇。然后，按照协议执行步骤1.5 - 1.7。添加5μl的细菌悬浮液在载玻片上并覆盖盖玻片。图像样本作为协议的步骤1.9。在这些条件下，人口的100％应是碘化丙啶阳性（ 图2）。在某些细菌物种，碘化丙啶未必完全压倒SYTO9染色，并不能存活的细菌可能会出现黄色或橙色。 </li><li>对吞噬细胞如嗜中性粒细胞，暴露细胞异丙醇-杀死的细菌，并确保该胞内细菌的100％的碘化丙啶阳性（ 图3）。对于非吞噬细胞，可能不内化死的细菌，它可以是足以治疗感染的细胞中与叠氮化钠或其它渗透细胞的抗微生物剂添加的活/死染色溶液之前。 </li></ol></li></ol> 2。细菌性评估与可行性格力SYTOXn和DAPI <ol><li>与10微克/毫升的DAPI 20分钟，在室温下在黑暗中在莫尔斯的定义中25兴趣标签细菌。 </li><li>感染的细胞，粘附到12毫米直径的圆形盖玻片在24孔板用DAPI标记的细菌。 不修复的细胞与醛或有机溶剂。 </li><li>与MOPS / 氯化镁冲洗一次细胞。 </li><li>孵育细胞10分钟，在室温下在黑暗中的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，在MOPS / MgCl 2的，以检测外部细菌。见注在协议的步骤1.3中建议的控制。 </li><li>从细胞中吸媒体和加入0.5毫升0.4微米SYTOX格林在MOPS / 氯化镁 。 </li><li>孵育细胞5分钟，在室温下在黑暗中。 </li><li>冲洗细胞两次在公开资讯观测站/ 氯化镁 。 </li><li>在MOPS / 氯化镁 5分钟洗涤细胞一次。 </li><li&gt;采集用荧光显微镜在30分钟内的图像。看显微镜，数码相机和采集软件描述协议的步骤1.9。 </li></ol>注意：使用过滤器具有590激发波长检测到的Alexa Fluor 647荧光 - 650 nm和663发射滤光片 - 735纳米，是假色红。 495 nm和515发射滤光片 - - 555 nm处使用过滤器具有465激发波长检测从SYTOX绿色荧光。 375 nm和400 nm的屏障过滤器 - 使用过滤器以355激发波长检测来自DAPI的荧光。 <ol start="10"><li>该协议将导致在外部不能存活的细菌出现红光+绿光图像，内部自生能力的细菌出现绿+蓝，外部活菌出现红+蓝，和内部活菌出现蓝色只（ 图4）。作为协议的输注液定量描述外部和内部的细菌的百分比p 1.11。 </li><li>在协议步骤1.12中描述的控制应与DAPI / SYTOX Green染料组合（ 图2和图3）来执行。 </li></ol> 3。评估细菌生存能力除了亚细胞定位<ol><li>在莫尔斯的定义中20分钟，在室温下在黑暗中有10微克/毫升的DAPI兴趣标签细菌。 </li><li>感染的细胞，粘附到12毫米直径的圆形盖玻片在24孔板用DAPI标记的细菌。 不修复的细胞与醛或有机溶剂。 </li><li>与MOPS / 氯化镁冲洗一次细胞。 </li><li>孵育10分钟，在室温下在黑暗中的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，在MOPS / MgCl 2的，以检测外部细菌。见注在协议的步骤1.3中建议的控制。 </li><li>从细胞中吸媒体和冲洗细胞2 TIMES在MOPS / 氯化镁。 </li><li>培养细胞的Alexa Fluor针对感兴趣的亚细胞标记物555偶联抗体20分钟，在MOPS / MgCl 2的含有0.2％皂苷。 </li><li>冲洗细胞两次在公开资讯观测站/ 氯化镁 。 </li><li>在MOPS / 氯化镁 5分钟洗涤细胞一次。 </li><li>从细胞中吸媒体，并添加0.4微米SYTOX格林在MOPS / 氯化镁 。 </li><li>孵育细胞5分钟，在室温下在黑暗中。 </li><li>冲洗细胞两次在公开资讯观测站/ 氯化镁 。 </li><li>在MOPS / 氯化镁 5分钟洗涤细胞一次。 </li><li>在荧光显微镜30分钟获得的幻灯片图像。看显微镜，数码相机和采集软件描述协议的步骤1.9。 </li></ol>注意：使用过滤器具有590激发波长检测到的Alexa Fluor 647荧光 - 650 nm和663发射滤光片 - 735纳米，是假色紫色。的荧光F580纳米和600的发射滤波器 - - 660纳米的ROM的Alexa Fluor 555使用滤波器540的激发波长进行检测。 495 nm和515发射滤光片 - - 555 nm处使用过滤器具有465激发波长检测从SYTOX绿色荧光。 375 nm和400 nm的屏障过滤器 - 使用过滤器以355激发波长检测来自DAPI的荧光。 <ol start="14"><li>该协议将导致在外部不能存活的细菌出现紫色+绿色的图像，内部自生能力的细菌出现绿+蓝，外部活菌出现紫色+蓝色，内生菌只出现蓝色，和亚细胞蛋白质呈红色（ 图4）。量化外部和内部活菌如协议中描述的perecent步骤1.11。 </li><li>在协议步骤1.12中描述的控制应与DAPI / SYTOX Green染料组合（ 图2和图3）来执行。 </li><li>在除了计数可行的和无法存活的细菌，细菌每一个分类为正或负的共定位与所关注的亚细胞标记。 </li><li>计算除以共同定位的存活细菌的数量通过活菌总数共定位与亚细胞标记物的存活细菌的百分比。计算除以无活力共定位的细菌数量由无活力的细菌总数共定位与亚细胞标记物不能存活的细菌的百分比。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

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

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。

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 ...

fluorescence-microscopy-methods-for-determining-viability-bacteria

Research

JoVE Journal

Immunology and Infection

43.5K 次观看.  University of Virginia Health Sciences Center. 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。 

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

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

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

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

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

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

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

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

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

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 ...

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

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

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

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

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

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic 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

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

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

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

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

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

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

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing 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 ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...