Name: monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in streptococcus pneumoniae using fluorescent dyes
Uploaded: 2014-02-17T22:30:00
Description: Watch this Scientific Journal Video about Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes at JoVE.c

这项工作是由比尔和梅琳达·盖茨基金会（批准53085），在JR Oishei基金会和美国肺脏协会（批准RG-123721-N）以APH和美国国立卫生研究院（NIDCD）奖学金F31DC011218选管会的支持。

1。准备细菌培养<ol><li> 用于实验的生长培养 <ol><li>在37℃下加热块，解冻冷冻的储存小瓶的S。肺炎和其内容添加到9毫升新鲜，预热的托德-Hewitt肉汤0.5％酵母提取物（THY），用于在玻璃培养管10ml的总体积。 </li><li>孵育静态地在37℃，直到培养物达到对数中期（600nm的绝对值≈0.5-0.6）。 </li></ol></li></ol> 2。检测膜去极化和破裂<ol><li> 试剂的准?...

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尽管该大小呈现为使用传统电生理学方法来检测变化的极性和膜和变化在细菌内的离子浓度的完整性的限制，我们已经描述了一种方法来测量在S.这些事件肺炎用荧光染料。我们的协议是首个同类型的肺炎球菌和几个一般描述为细菌的物种之一描述。通过使用荧光检测板读数器，这些事件可以在小的，200微升体积的细菌的样品进行测量，以从细菌随时间的个体群中检测到的荧光。动?...

我们实验室已经确定了从名为哈姆雷特人乳（适用于人α-乳清蛋白制成致命的肿瘤细胞）诱导肿瘤细胞凋亡的蛋白-脂质复合物，但也能杀死多种细菌种类1,2。被发现是特别敏感的物种是那些靶向呼吸道， 肺炎链球菌 （肺炎球菌）显示的最大灵敏度和死亡2,3的凋亡样表型。膜去极化和特定的离子迁移的事件是充分描述并在真核细胞中，特别是在线粒体中，其中放射性TPP +离子和荧光染料包括JC-1和TMRE已被用于证明线粒体膜3的去极化的细胞凋亡过程中的关键事件-5。因此，我们试图了解更多关于哈姆雷特对这些膜相关的功能在肺炎球菌的影响，因为我们集中我们的努力，开发更好地理解本凋亡样表型中的细菌，与用于识别的过程中新抗菌疗法或疫苗候选物的巨大潜力的机械部件。 在寻 ​​求建立的协议为我们机理研究，我们发现，相对于在真核系统的良好描述的方法，也有极少数已发表 ​​的研究详细介绍了细菌膜6,7的电生理和离子传输机制。这主要归因于微生物的较小的尺寸和它们的表面架构，尤其是细胞壁的存在，这限制了薄膜的使用常规真核的方法，如膜片钳的可达性，尽管使用巨型原生质一些研究已经混合成功执行8,9。作为与这些巨型原生质体的工作是不是大多数细菌物种的理想甚或实用的方法，因为它需要人ipulated细菌在一个不自然的和非生物的状态下，细菌细胞膜极性已经执行了有限的研究主要采用流式细胞术和使用花青和氧杂菁荧光染料10-16。 代替的流式细胞仪，其集个别荧光读数从一种细菌在单一时间点上，我们选择使用荧光检测板读取器来检测细菌悬浮液的荧光强度随时间的96孔板形式。这使我们能够把细菌人口在各时间点与更大的简便和易用性，并持续监测整个人口的荧光动力学的时间延长周期，这是很难实现的使用流式细胞仪。测试各种潜在的敏感的荧光染料（包括上述与线粒体利用提及）之后，我们实现了用最好的技术和实际成功双-氧杂菁染料称为DIBAC 4（3）（双- （1,3 - dibutylbarbituric酸）三甲氧杂菁）来监视改变极性。 我们还发现它有价值使用碘化丙啶（PI）的同时监测该膜的完整性的破坏。这种染料发出荧光结合后的核酸，但只能够这样做，当细菌细胞膜的完整性受到损害，使得它用于检测死细胞在活死染色法测定的流行成分。除了​​PI，SYTOX绿色和TO-PRO-1的荧光染料是在行动相似，先前已被用于细菌利用流式细胞仪检测方法17几个研究。我们选择使用PI由于其激发波长，使我们能够同时监测其荧光与DIBAC一个给定的样本。 在我们的研究中，我们已经观察到小村庄，以及另一个相关的蛋白 - 脂质复合物与杀菌活性通过增加两种染料的处理后的肺炎球菌3,18,26的荧光所指示的被称为ELOA，诱发的去极化细菌细胞膜的破裂。对于这两种复合物中，我们观察到，DIBAC 4的荧光强度（3）增加的PI的强度之前增加，表明破裂之前去极化发生的，因此，引起了杀菌蛋白质-类脂复合体的一个特定的事件兴趣。这种区别是重要的，以作为该膜的破裂本身可引起非特异性的去极化。测量和分析都DIBAC 4（3）和PI荧光动力学同时允许我们研究这两种膜的事件，这是用荧光代替流式细胞仪的一个附加的优点之间的这种关系。 为了监测细菌的离子流，出现了一些以前的成功使用放射性同位素，包括测量的摄取45的Ca 2 +中的肺炎球菌19,20，我们也用在我们最近的研究18，21。然而，与这些放射性离子加工有几个缺点。它们可以是昂贵的，耗时的，并且杂乱，并且还可使本个体进行实验伤害，这取决于所感兴趣的同位素。此外，也很难随着时间的推移监视的快速变化。因此，我们转向测量的另一种方法，它采用的离子敏感的荧光指示剂染料乙酰氧甲基（AM）酯版本。由本身的指示剂染料被充电，并通过该膜不容易通过，但增加了亲脂性酯基团的，现在不带电荷的分子能够穿过细菌的膜。一旦进入室内，细菌酯酶裂解酯基，使染料自由在细胞内，并再次充电，显著减缓其能力，退出电池和使染料吨Ø内部积累随着时间的推移。然而，使用这些酯染料只被在几个细菌种类中描述的变化来检测细胞内Ca 2 + 22-24和H + 16，具有不同的负载，检测，和成功的方法。 用监视改变细胞内钙欲望2 +，也K +和H +的水平在S.因肺炎与哈姆雷特和其他化合物的治疗，我们成功创建协议来高效地加载荧光指示剂染料进入细菌细胞。有效地加载到所需的两个丙磺舒，通过阻断阴离子转运和电力负荷，自Life Technologies专有的化合物，它提高了装载效率提高了染料滞留细菌。 Fura-2/AM荧光（检测的Ca 2 +），PBFI / AM（检测K +），和BCECF / AM（检测H +）被成功地装入两个未封装和包封的肺炎球菌ST降雨使所得到的荧光图案的测定除了离子载体，例如离子霉素（Ca 2 +的解偶联剂），缬氨霉素（K +解偶联剂）和CCCP（H +解偶联剂）使用荧光检测板阅读器18，21的后。

<table border="0" cellpadding="0" cellspacing="0" width="864">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Todd Hewitt</td>
			<td style="width:139px;">Bacto, BD Diagnostics</td>
			<td style="width:143px;">249240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Yeast Extract</td>
			<td style="width:139px;">Bacto, BD Diagnostics</td>
			<td style="width:143px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Phosphate Buffered Saline (PBS; pH 7.2)</td>
			<td style="width:139px;">Invitrogen (GIBCO)</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Dimethyl sulfoxide</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">D5879</td>
			<td>DMSO</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:359px;">DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol)</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">B-438</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Propidium Iodide</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4170</td>
			<td>Make up in deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-(+)-Glucose</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PowerLoad</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P10020</td>
			<td>100X concentrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Probenecid</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P36400</td>
			<td>Make 100X stock by adding 1 ml of PBS to one 77 mg vial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Fura-2/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">F1221</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">PBFI/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">P1267</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Nigericin</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;">N7143</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">3246-01</td>
			<td>monobasic</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">K2HPO4</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">4012-01</td>
			<td>dibasic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">NaOH</td>
			<td style="width:139px;">JT Baker</td>
			<td style="width:143px;">5565-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">BCECF/AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:143px;">B1170</td>
			<td>Special packaging (50 &#181;g aliquots)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">CCCP</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:143px;"></td>
			<td>Protonophore that causes an influx of H+ into the cytoplasm,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>&#160;dissipating the electrical potential and the H+ gradient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Culture tube&#160;</td>
			<td style="width:139px;">VWR</td>
			<td style="width:143px;">53283-802</td>
			<td>Fits the Spectronic spectrophotometer; borosilicate glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Spectrophotometer</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:143px;"></td>
			<td>Spectronic 20D+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">15 ml plastic conical tube</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Clear 96-well polystyrene microtiter plate</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-565-501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Plate reader</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:143px;">Synergy 2 Multi-Mode&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;"></td>
			<td style="width:139px;"></td>
			<td style="width:143px;">Microplate Reader</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Gen5 software</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:143px;">Gen5&#8482; Software</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in streptococcus pneumoniae using fluorescent dyes

膜去极化和离子通量是已被广泛地研究在生物系统中，由于他们的能力，深刻地影响细胞功能，包括能量和信号转导事件。虽然这两种荧光及电生理方法，包括使用电极和膜片钳，得到了很好的发展，用于测量在真核细胞中这些事件，方法用于测量微生物的类似事件已被证明更具挑战性的发展给它们的小尺寸与更复杂的组合细菌屏蔽膜的外表面。在我们的死亡引发的肺炎链球菌 （肺炎球菌）的研究，我们想阐明膜事件，包括改变极性，完整性和细胞内离子浓度的作用。检索文献，我们发现，很少有研究存在的。其他调查人员监控放射性同位素的吸收或平衡测量离子感XES和膜电位和研究，大多是在革兰氏阴性菌的数量有限，使用碳菁或氧杂菁染料的荧光来测量膜电位，或加载与细胞穿透物的乙酰氧甲基（AM）酯版本细菌已经看到了一些成功离子敏感荧光指示剂染料。因此，我们建立和优化的协议，用于测量在革兰氏阳性生物体S.膜电位，破裂，和离子转运肺炎 ， 我们开发了使用双-氧杂菁染料DIBAC 4（3）和小区浸透染料碘化丙啶来测量膜去极化和破裂，分别，以及方法，以最佳地加载肺炎球菌的比例染料的AM酯协议Fura-2的，PBFI，BCECF和变化来检测细胞内浓度的Ca 2 +，K +和H分别+的，使用荧光检测用平板阅读器。这些协议是首个同类型的肺炎球菌与大多数这些染料没有被用在任何其他细菌物种。虽然我们的协议进行了优化，S。肺炎 ，我们相信这些方法应该形成一个很好的起点在其他细菌物种类似的研究。

对于所有的实验中，有一个样品和一组存在于每个孔中的条件。因此，每个跟踪指细菌随着时间的整个群体的荧光强度。结果应该是容易解释的，用处理后的样品和未处理对照的荧光有明显区别。在荧光的观察到的变化的动力学和程度可提供约被监视的事件的可能机制及程度。 当探索膜的极性，细菌必须孵育DIBAC约40分钟，以允许染料的平衡过的膜，通过在处理之前的?...

Watch this Scientific Journal Video about Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes at JoVE.c

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

不像见过的真核生物中，有研究贫乏，在细菌细节膜去极化和离子浓度的变化，主要是由于它们的小尺寸使得难以测量的常规方法。在这里，我们监测的显著革兰氏阳性病原体肺炎链球菌利用荧光技术这样的事件细节的协议。

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Research

JoVE Journal

Immunology and Infection

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

纯化膜隔间跨演示文稿中的外源抗原内质网关联降解

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

双光子激光显微镜在细胞膜修复中的应用

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

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 ...