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> 试剂的准备 <ol><li>制备（3）在100％DMSO中的50μM的库存DIBAC 4和2毫克/毫升的股票的PI在DDH 2 O。该DIBAC 4（3）库存稳定在-20℃下至少6个月。 PI的股票是稳定在4℃下至少6个月。 </li><li>个股包裹在铝箔从光保护。 </li></ol></li><li> 载入中菌<ol><li>通过离心分离沉淀从步骤1.2.2细菌细胞在2400×g下在室温下10分钟，并除去上清液在2400×g离心洗涤两次，重悬沉淀在10ml的1X磷酸盐缓冲盐水（PBS）中，并离心分离机的再持续10分钟。 </li><li>去除上清，重悬沉淀在PBS到原来的体积。这将提供约10 8个菌落形成每毫升细菌的单元。除去1毫升（足够用于5个样本）的洗涤细胞并置于微量离心管中。 </li><li>到这些细胞中，为25mM的葡萄糖的终浓度添加25微升的葡萄糖的1M过滤灭菌的溶液（制备在DDH 2 O，并通过0.45μm的过滤器过滤），并孵育在37℃的加热块十五分钟。注：提供的能量为所有依赖ATP的膜通道和细胞成分。需要这个步骤可能会有所不同，这取决于实验。 </li><li>加入5微升的50微米DIBAC 4（3）股票和10μl的2毫克/毫升PI股票。这提供了为250nm的最终浓度和20微克/毫升。移液器向上和向下几次调匀。添加200μl的该细胞悬浮液，以明确的96孔板的每个样品孔。 </li></ol></li><li> 检测荧光 <ol><li>现浇板在预热（37℃）荧光检测酶标仪。 </li><li>确保适当的过滤器，让本DIBAC 4（3）（490 nm激发，516 nm发射）和PI（535 nm激发，617 nm发射）检测。 </li><li>为了让（兼监察）DIBAC 4的平衡（3）在膜，设置读者采取荧光测量每分钟约30-40分钟，或直至读数稳定，这作为一个“预处理”的阅读。 </li><li>退出平板并添加选择的实验药物和立即数iately将板放回读者继续监测DIBAC 4（3）和PI荧光的时间所需要的长度。如果处理多个样品一次，用多通道移液器可以是有帮助的同时添加剂到所有孔中。 </li><li>导出读数到一个数据分析程序，如Microsoft Excel中。情节荧光随着时间的推移，以评估膜去极化和破裂。 DIBAC和PI的荧光增加表明，除极和破裂正在发生。 </li></ol></li></ol> 3。检测改变细胞内的Ca 2 +或K +浓度<ol><li> 准备试剂和上样缓冲液 <ol><li>制备以下试剂：对离子敏感的荧光染料的5mM储备（Fura-2/AM荧光或PBFI / AM;加DMSO的适量含50μg染料的管），“100X”丙磺舒（加1毫升的PBS到77毫克小瓶）。溶解的染料可在-20℃一次冻结并稳定在3个月的溶液，而丙磺舒是稳定的溶液在-20℃下6个月。 </li><li>准备加入1ml 2×上样缓冲液如下：在一个15毫升的塑料锥形管的底部，取20μl的100倍型电力浓缩物（稳定在2-8℃下进行6个月），随后加入2微升的5mM的离子的敏感染料直接插入型电力。轻轻振荡混匀。添加960微升PBS接着20μl的100倍丙磺舒。旋涡混合，最后一次。包装在铝箔避光。 </li></ol></li><li> 中的细菌与染料 <ol><li>沉淀并如2.2.1中所述洗涤中对数期细菌培养。除去上清，重悬沉淀在5ml PBS中，使一“2×”细胞悬浮液。 </li><li>加入1 ml的2倍的细胞悬浮于2×上样缓冲液。这将提供2毫升的最终体积，并最终concentr1X型电力，5微米的荧光染料，和1x丙磺舒ations。注：此悬挂提供了足够的容量为10的样本，因为每个样品以及测量将包含200微升。 </li><li>孵育在37℃水浴75分钟，避光。注：该染料被加载到细菌细胞中不存在任何糖基片和在丙磺舒，以尽量减少染料的流出的存在。 </li><li>洗1：颗粒的细胞通过离心在2400×g离心10分钟，除去上清液，并含有1x丙磺舒重新悬浮于2ml的PBS中。洗2，3：重复步骤3.2.4 2倍。在37℃下30分钟。这将允许时间为细菌酯酶水解的AM酯基团远离内在荧光染料。 </li><li> WASH 4：颗粒细胞离心在2400×g离心10分钟，弃上清，重悬在2毫升的PBS中含有1x丙磺舒。注：根据不同的experimeNT，葡萄糖可以加回到这里重新注入活力的细菌。 </li></ol></li><li> 检测荧光 <ol><li> Prewarm荧光检测板阅读器读板器至37℃。 </li><li>对于每个所需的样品，加入200微升的负载细胞（来自步骤3.2.8）的一个96孔板的孔中。 （请执行下列步骤来完成一个井/采样一次）。 </li><li>为了建立基线的荧光检测板阅读器测量荧光（340 nm激发/ 510 nm发射的“值1”和380 nm的激发/ 510 nm发射的“值2”）在读的很好，地方板每一秒为1分钟。 </li><li>弹出板，加选择的治疗那么好（在小体积的约5微升），迅速吹吸几次混合，并立即放回在荧光检测板阅读器读取每个秒的期望长的时间。 （如果在可与平板阅读器相关联的jectors，这些也可以用于无缝地添加所希望的处理，从而无需停止读和弹出该盘）。 </li><li>导出读数到一个数据分析程序，如Microsoft Excel中。计算每个时间点的荧光值的比率除以值1由值2，并绘制在图上的比例值。 </li></ol></li></ol> 4。检测改变细胞内pH值<ol><li> 准备试剂和校准缓冲液 <ol><li>准备下列试剂：BCECF / AM 5毫米的股票（。添加二甲基亚砜适量含50微克染料的管这个股票是稳定在-20°C为3个月）; 100X丙磺舒（加入1毫升PBS为77毫克小瓶）; 1毫米的股票在乙醇制备尼日利亚菌素的细胞周围的缓冲液的pH为（以平衡细胞内pH值（pH值我需要的）这个股票是S表在-20℃下6个月）;。羰基氰-3 - 氯苯（CCCP）在DMSO（protonophore，以便拆开质子动力的0.5毫米的股票该股票是稳定在-20℃下6个月） </li><li>准备好10毫升以下含钾高的缓冲液：135 mM的KH 2 PO 4/20毫米氢氧化钠（一元）和110毫米的K 2 HPO 4/20毫米氢氧化钠（二元）。过滤消毒用0.45微米的过滤器。 （这些缓冲区可以被保存在4℃直至使用）。 </li><li>混合高钾缓冲在必要的比例来创建高钾缓冲至少有5-6的pH值范围为6.5〜8.0。 </li><li>准备加入1ml 2×上样缓冲液如下：在一个15毫升的塑料锥形管的底部，取40微升100倍型电力，接着将20μl的5mM离子敏感染料直接插入型电力。轻轻振荡混匀。添加900微升PBS随后加入40μl的100倍丙磺舒。 Vort恩，最后一次混合物。包装在铝箔避光。 </li></ol></li><li> 中的细菌与染料 <ol><li>沉淀和洗涤8毫升的半对数期细菌培养物如在2.2.1中描述。去除上清，重悬沉淀在2毫升的PBS做一个“4倍”细胞悬液。 </li><li>加入1 ml的4倍的细胞悬浮于2×上样缓冲液。这将提供2毫升的最终体积，并终浓度2倍的细胞，2型电力，50μM的荧光染料，和2x丙磺舒。 </li><li>孵育在30℃水浴40分钟，避光。注：染料被装入细菌细胞在30℃的较低温度，并在没有任何糖酵解底物，以减少染料的流出。 </li><li> WASH 1：颗粒细胞离心在2400×g离心10分钟，弃上清除掉多余的染料，以及含有2倍丙磺舒一个重悬在4毫升的PBSND 1毫米的葡萄糖（恢复活力的细菌）。洗2，3：重复步骤4.2.7两次，在4毫升PBS中再悬浮最终（用于1X细胞的最终浓度）含有2个丙磺舒和10mM葡萄糖。 </li><li>在37℃下5分钟。这将允许在细胞充分激励和时间让细菌酯酶裂解AM酯基离内在BCECF。 </li></ol></li><li> 建立背景荧光和体内的校准曲线 <ol><li> Prewarm荧光检测板阅读器读板器至37℃。 </li><li>以确定背景荧光，过滤灭菌约500微升的加载和通电细菌悬浮液（来自步骤4.2.7）以除去细菌，并添加200μl的滤液到96孔板的孔中。 </li><li>将板在荧光检测板阅读器测量荧光（490 nm激发/ 530 nm发射的“值1”和1分钟440 nm激发/ 530 nm发射的“值2”）。此背景荧光应为校准曲线和实验样品的比率的计算之前被减去。 </li><li>为了获得在体内校准曲线，在高钾缓冲一次，重悬洗加载的，充满活力的细胞500μL分装步骤4.2.7在不同pH值（范围从6.5-8.0）。 </li><li>添加20μM的尼日利亚菌素对样品平衡的细胞的细胞内pH值到周围的缓冲液的pH和孵育在37℃下5分钟。加入200μl的细菌/ pH缓冲组合到一个96孔板的孔中。 </li><li>将板在荧光检测板阅读器测量荧光几分钟来建立一个稳定的读数为每口井。导出读数到一个数据分析程序，如Microsoft Excel中。 </li><li>减去BA后ckground荧光值，除以价值1由Value 2计算出荧光值的比值。绘制每个pH缓冲剂的比例值来创建校正曲线。 （应为每一批新加载的细胞中产生的校正曲线，作为被装入细菌荧光染料的量可以变化，每次）。 </li></ol></li><li> 在细胞内pH测量的变化 <ol><li>对于每个所需的样品，加入200微升的加载和通电细胞（来自步骤4.2.7）的一个96孔板的孔中。 </li><li>放置板中的荧光检测板阅读器，并测量样品的荧光，每隔5秒5分钟。 </li><li>喷射板，并添加10μMCCCP到一个阱（作为阳性对照，以减少细胞内pH）和感兴趣的其它井的试验剂（次）。为了准确地比较不同时间的效果，添加了CCCP和选用的药物同时使用时ultichannel吸管。 </li><li>立即将板返回到板阅读器，并继续测量荧光每5秒10分钟。作为一个额外的控制，喷射板，并添加20μM尼日利亚菌素对所有样品达到平衡，细菌的pH值i到周围的缓冲液的pH，并读5分钟。 </li><li>减去背景荧光值后，计算荧光各样品的比率。插pH值i表示每个读数从校正曲线。 </li></ol></li></ol>

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Chem. 287 (32), 27168-27182 (2012).</li><li>Trombe, M. C., Laneelle, G., Sicard, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Streptococcus+pneumoniae+mutant+with+altered+electric+transmembrane+potential.">Characterization of a Streptococcus pneumoniae mutant with altered electric transmembrane potential.</a> J. Bacteriol. 158 (3), 1109-1114 (1984).</li><li>Trombe, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+calcium+porter+of+Streptococcus+pneumoniae+involved+in+calcium+regulation+of+growth+and+competence.">Characterization of a calcium porter of Streptococcus pneumoniae involved in calcium regulation of growth and competence.</a> J. Gen. Microbiol. 139 (3), 433-439 (1993).</li><li>Marks, L. R., Clementi, E. A., Hakansson, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitization+of+Staphylococcus+aureus+to+Methicillin+and+Other+Antibiotics+In+Vitro+and+In+Vivo+in+the+Presence+of+HAMLET.">Sensitization of Staphylococcus aureus to Methicillin and Other Antibiotics In Vitro and In Vivo in the Presence of HAMLET.</a> PLoS ONE. 8 (5), (2013).</li><li>Futsaether, C. M., Johnsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fura-2+to+measure+intracellular+free+calcium+in+Propionibacterium+acnes.">Using fura-2 to measure intracellular free calcium in Propionibacterium acnes.</a> Can. J. Microbiol. 40 (6), 439-445 (1994).</li><li>Tisa, L. S., Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+properties+of+Escherichia+coli+mutants+having+abnormal+Ca2++content.">Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content.</a> J. Bacteriol. 177 (24), 7112-7118 (1995).</li><li>Werthen, M., Lundgren, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++mobilization+and+kinase+activity+during+acylated+homoserine+lactone-dependent+quorum+sensing+in+Serratia+liquefaciens.">Intracellular Ca2+ mobilization and kinase activity during acylated homoserine lactone-dependent quorum sensing in Serratia liquefaciens.</a> J. Biol. Chem. 276 (9), 6468-6472 (2001).</li><li>Avery, O. T., MacLeod, C. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Complex+of+Equine+Lysozyme+and+Oleic+Acid+with+Bactericidal+Activity+against.">A Complex of Equine Lysozyme and Oleic Acid with Bactericidal Activity against.</a> PLoS One. . 8, (2013).</li></ol>

作者没有竞争经济利益申报。

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 ...

monitoring-changes-membrane-polarity-membrane-integrity-intracellular

Research

JoVE Journal

Immunology and Infection

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

13.5K 次观看.  University at Buffalo, State University of New York. 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。 

视频: 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

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

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

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

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

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

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

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

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

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

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

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

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

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

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

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

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

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

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

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

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

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation 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

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

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

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 ...