Supported by NIEHS grants #R01 ES013268-01A2, and the contents are solely the responsibility of the author and do not necessarily represent the official views of the NIEHS, and supported by CETOCOEN UPgrade project No. CZ.1.05/2.1.00/19.0382.

这项研究的方案经动物护理和日本健康科学，这就是体内都做过实验，全国学院利用委员会，以确保将大鼠人道和对苦难的缓解方面的治疗。 1. SL-DT生物测定<ol><li>种子3×10 5个 WB-F344大鼠肝脏上皮细胞到35 mm直径培养含有鹰板改性介质加5％牛胎儿血清，并培养所述细胞在培养箱中在37ºC，100％相对湿度（RH），5％ 的 CO 2。 </li><li>培养细胞，直到它们达到100％汇合（通常为2天）和如下所述进行细胞的期望实验性治疗。 <ol><li>进行剂量响应实验<ol><li>添加10微升和20微升菲的在100％乙腈和4微升，6微升2mM的储备液和8微升phenan的20mM储备溶液的士林在100％乙腈到含2ml生长培养基为原液每个添加体积细胞的三个板。 </li><li>加入4微升，6微升，8微升，10微升，和20μl乙腈的100％的溶液，以含2ml生长培养基为乙腈溶液各自加入量作为车辆控制单元的三个板的。 </li><li>孵育15分钟，板在设定为37℃，100％相对湿度和5％的CO 2的培养箱。 </li><li>继续执行步骤1.3。 </li></ol></li><li>开展时间响应实验<ol><li>加7微升菲的在100％乙腈的20mM储备溶液到含2ml生长培养基的每个时间点（ 即 0，1，2，3，4，5，10分钟）细胞的三个板。 </li><li>加7微升100％乙腈到含2ml生长培养基的每个时间点，作为车辆控制单元的三个板。 </li><li>孵育每个指定时间p内板oint在培养箱中在37℃，100％相对湿度和5％ 的 CO 2。 </li><li>继续执行步骤1.3。 </li></ol></li><li>开展时间恢复实验<ol><li>加7微升菲的在100％乙腈的20mM储备溶液到含2ml生长培养基的15分钟的细胞的三个板。 </li><li>加7微升100％乙腈到含2ml生长培养基的15分钟，作为车辆控制单元的三个板。 </li><li>倒出含有任菲或乙腈中，每个冲洗3次用3ml磷酸盐缓冲盐水（PBS）中。 </li><li> 2毫升新鲜生长培养基添加到每个板并孵育所需的恢复时间（ 即，25，35，45，60，90，120，150，180，240，360分钟），在37℃的恒温箱中，100 ％RH下和5％ 的 CO 2。 </li><li>继续执行步骤1.3。 </li></ol></li><li>传导机制实验<ol><li>加的信号传导的抑制剂， 即 </em&gt;，5微升D609的在100％乙腈中的20mM储备溶液到含2ml生长培养基的15分钟的细胞的三个板的。 </li><li>添加5μl的100％乙腈到含2ml生长培养基的15分钟，作为车辆控制单元的三个板。 </li><li>加7微升菲的在100％乙腈的20mM储备溶液到含2ml生长培养基加上D609的细胞的三个板另外15分钟。 </li><li>加7微升100％乙腈到含2ml生长培养基加上100％乙腈的细胞的三个板另外15分钟，以作为该车辆的控制。 </li><li>继续执行步骤1.3。 </li></ol></li></ol></li><li>由任一轻轻倾倒出培养基或通过真空吸附丢弃培养基。 注:适当处置含有危险废物的培养基。 </li><li>冲洗细胞三次，每次用3ml PBS中，要么澄清或抽吸betweeñ冲洗。 </li><li>吸管1毫升1毫克/毫升萤光黄染料溶解在PBS（LY）到每个单元板。 </li><li>通过细胞群体中的板的三个不同区域轻轻滚动＃20外科钢叶片具有圆形边缘预加载染料进入细胞。 <ol><li>通过将垂直的手术刀（90°角），并从培养板的边缘5毫米开始，然后转离这个垂直的角度手术刀至约15°（ 见图1）的角度。 注:这是通过捏拇指和食指之间的手术刀来实现的。利用重力让手术刀轻轻地从90°到15°的位置（如圆形刀片只需鼠标滑过细胞）。这项议案将留下一个视觉明显的压痕线。 </li></ol></li><li>孵育所述细胞在室温下的LY溶液3分钟。 </li><li>倒出或者吸出LY，并用3毫升的PBS以除去所有extrace冲洗三次llular染料消除外背景荧光。 </li><li>加入0.5毫升10％磷酸盐缓冲的福尔马林溶液来固定细胞。 注意:在福尔马林固定后，空气干燥的细胞并在黑暗中保存长达两年的LY染料的最小光漂白。当需要时，补充水分，用10％的福尔马林溶液来可视化的荧光染料前。 </li><li>查看使用在200X的放大倍数配备了分色立方体的五百三十六分之四百二十八激发/发射峰的落射荧光显微镜的定格。对齐所有板，使压痕线平行视力水平场。 注:FITC二向色立方体效果很好。 <ol><li>与CCD相机和支持软件捕获图像。 <ol><li>打开CCD摄像机的配套软件，并使用设置自动曝光。使用"捕获"按钮，以数字方式获取图像。将图像保存为.TIF使用"保存"按钮的文件。 </li></o升&gt; </li></ol></li></ol> 2.适应的SL-DT检测到肝组织<ol><li>从5周龄，用雄性菲舍尔344大鼠解剖剪刀取出大约从肝左叶一2×2cm的片，将其放在一个塑料板掂量盖上湿纱布12。 </li><li>吸管0.5毫升含有1mg的PBS溶液/ ml的各荧光黄和罗丹明 - 葡聚糖（RD）上的肝切片的在塑料表面称量板。 注意:RD为不能穿过的间隙连接通道，将标记加载的细胞的染料。 </li><li>使三至五个切口肝脏切片具有用锋利的刀片在称重盘的染料溶液的表面上长大约1cm，然后添加足以填充切口附加染料。 </li><li>孵育所述组织在室温下在塑料称量板3分钟。 </li><li>用5毫升的PBS冲洗三次。 </li><li>修复组织奥雅纳rnight在黑暗中在室温下在50ml锥形离心管30含10％磷酸盐缓冲的福尔马林。 </li><li>次日，洗涤片，用水，修剪组织切口周围用解剖剪成1×1×0.5 cm 3的条带，然后使用标准技术来嵌入切片的石蜡13。 </li><li>部分使用标准切片技术用切片机13被成像在垂直于切口线5微米厚的切片，并存储样品在黑暗中直到准备。 </li><li>使用配有CCD数码相机按照第7 1.10可视化和捕捉荧光染料方面的图像的落射荧光显微镜。 </li></ol> 3.量化GJIC <ol><li>使用形态的软件包（ 例如 ，ImageJ的）测量荧光染料扩散。 <ol><li>点击"文件"选项卡，然后单击"打开"，打开保存的图像。 </li><li>点击"免费手工具"，在工具栏选项卡来跟踪染料前端的轮廓。 </li><li>单击"分析"选项卡，然后单击"测量"。 注意:这将产生在一个扩散片，其如果需要，可以复制到其它电子数据表程序的区域的值。 </li></ol></li><li>使用电子表格程序，通过将在实验板通过使用以下公式中的控制板上的染料行进的区域中的染料扩散的面积计算控制（FOC）的分数: <img alt="公式1" src="/files/ftp_upload/54281/54281equation1.jpg" /> A E =在暴露于实验变量的细胞，例如用化学在特定剂量或时间处理的细胞中的染料扩散的区域 染料扩散的控制，这是与用于溶解所关注的化学品的载体处理的细胞的C =面积 注意:为了确定车辆的效果，A E将是染料扩散的区域中由车辆处理的细胞和C是在不通过车辆处理的细胞中的染料扩散的面积。车辆的效果应优选小于10％。 </li></ol>

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The authors have nothing to disclose.

对SL-DT测定是在测定GJIC简单和通用的技术，但也有要在设计适当的实验方案来解释几个关键的关注。对于使用SL-DT测定细胞间隙连接通讯的强大的测量必须有通过细胞间隙连接的LY良好的染料扩散。在最低限度，有足够的时间应选择以保证染料通过八个或更多列细胞扩展从两个方向的手术刀加载细胞。另外，为了便于将染料从手术刀装载的细胞迁移的距离的测量，选择确保了控制板的染料扩散填充视野的70〜90％的倍率因子。对于WB-F344大鼠肝上皮细胞，与LY解决方法三分钟培养足以使染料前到超过四级单元行移动，和200X的放大倍数是最佳的。细胞生长在35 mm直径板，这是相当易于进行loadi纳克染料成用手术刀细胞。然而，细胞可以在小水井中生长，但使用手术刀是不可能的，尤其是在96孔培养板中。对于小水井，用小圆头尖端，能适合入井，并再次，使用滚动操作。这种滚动操作是关键的一步，因为它是微创，并提供了一个非常干净的线负载，防止细胞被撕裂，创建典型的原始刮法大无细胞间隙。可选地，针针可以使用，其中所述针的小捻确实装载染料的一个很好的工作进入细胞。 其他细胞类型可以在此测定法中使用。但是，细胞的选择和理解GJIC的角色在特定细胞类型是在取景适当的假设和实验的设计很重要。第一个也是最明显的一步是开发细胞培养条件，使功能间隙连接的EXPRESSED。在分化良好的细胞类型，其执行特定的代谢功能，间隙连接通常在协调邻接小区之间的代谢途径的作用。良好分化的细胞类型具有有限，如果有的话，增殖能力和信道可能播放往往取决于形态和细胞的具体结构的组织特异性代谢作用。例如，心肌细胞在哪里间隙连接是在闰盘主要对准，从而使用SL-DT方法将需要对准的细胞，其可以在一个开槽盘来完成细长细胞。因此，仔细考虑必须为每个细胞类型进行。与神经元细胞培养物研究通常是不汇合并且还需要特定的接触点，这使得利用对SL-DT不适当和替代GJIC测定中必须使用。 在增殖的细胞类型，例如干细胞和早期祖细胞，GJIC起着更关键的作用在协调细胞信号控制基因表达调控的有丝分裂活动的事件。在这个协议中使用的WB-F344大鼠肝上皮细胞就属于这一类。连接蛋白基因是组织开发和维护19-21期间的关键。虽然间隙连接通道被表达并形成所有细胞质膜的表面上，增殖细胞对生长因子，其抑制GJIC非常敏感。用于增殖细胞类型的关键因素通常被定向更向媒体的组成。生长因子在过量常常是，特别是在含胎牛血清未定义培养基中，细胞的间隙连接可处于关闭状态。建立细胞间隙连接通讯在这些细胞中最简单的方法是减少或实验前取出4至24小时血清水平，但24小时将是细胞太久没有血清，因为他们将apoptose。用于实验的WB-F344肝上皮细胞在叔呈现他的论文都没有，没有必要减少血清血清过于敏感。与此相反，使用C10小鼠细胞系实验必须剥夺建立GJIC 22首先血清。虽然血清水平的两种细胞类型之间变化，有趣的是，C-10细胞反应非常相似的蒽22,23甲基异构体。 质量结果其他考虑因素包括在开展非细胞毒性剂量的实验。细胞需要用相差显微镜来确定前和治疗后的预期细胞类型的健康形态特征进行检查。为了确保在间隙连接功能的化合物的效果不是由于一般的细胞毒性反应，对于每一化合物的细胞毒性测定法应在同一时间进行，并在对SL-DT测定剂量使用。在进行实验，以确定是否对GJIC化合物的抑制效果是可逆的，也是至少有两个原因良好的实验。莫可逆测试ST化合物抑制GJIC，但如果没有的GJIC恢复，则细胞毒性可能是一个因素，并进一步测试将需要。然而，如果恢复GJIC则剂量和测试时间很可能没有细胞毒性。有用于GJIC的可逆抑制生物的影响。许多环境和食源性有毒物质和毒素的不利健康的影响是可逆的过程。因此，在分子水平上理解这种可逆性是重要的。如果未知化合物不可逆地抑制的GJIC，那么对于生物体的健康的影响可能是从其他大多数剂完全不同的，并且需要以风险计算的一部分。 确定参与GJIC的抑制的剂量和时间是一个反复的过程。为了节省时间和金钱为文化用品，最好的办法是开始在一个较高的剂量，这取决于溶解度和时间（通常为一个小时，因为大多数抑制发生在小于15分钟），然后再减少一半的时间和剂量，在步骤明智过程直到没有效果。如果在一个高剂量没有抑制在一小时后，加倍在步骤明智过程的时间段。最初的实验可以在单个板来完成。一旦通用时间估计和剂量被建立，然后更彻底的研究，可以有效地设计适当的样本大小的统计分析。一种实验性的设计特征是使用该达到75至100％抑制的剂量和时间。显然，结果最终的解释将上述参数的一个因素。然而，通过首先确定的生物效应，即使剂量和时间可能不体内相关，建立的剂量可以发挥职业暴露的情况下的作用，或暴露于其它的因素一般人群也影响GJIC与潜在添加剂或协同效应。 G的抑制JIC大多数化合物通常不是高度可变和两个到三个重复（每个重复将是一个培养板或孔）足以产生时间和剂量响应曲线。但是，如果变化是那么高，将需要更多的重复，以获取精确值一个独立审判的一个给定的因变量。当然，至少三个独立试验需要进行统计分析，以完成。 该测定与显著细胞群体大小的评估的简单性对于许多应用是有利的。这是在许多毒理学评估中特别有用。但是，也有对SL-DT测定法，其可以取决于若干因素的限制。一个是所研究的细胞这个技术是否是适当的。该测定不会为不与整个培养板或孔零星接触达到高汇合细胞很好地工作。替代技术，如微注射应共nsidered。可替代地，对SL-DT测定可以被修改。成功加载单个细胞已经实现了针灸针，加载染料7时，这需要一个轻微的扭转作用。染料的扩散是通过与零星的细胞接触的细胞培养更多的融合情况下的径向或无定形。被推荐用于实验中识别装载的细胞将是困难的未经标记（例如RD）加入罗丹明葡聚糖（RD，1毫克/毫升）至LY染料溶液。 RD太大遍历间隙连接通道，从而确定哪些加载细胞。 RD也可以在所有的SL-DT测定中使用，但将手术刀叶上的菜查明细胞加载的底部的凹口。在SL-DT法也是不利于不同类型的细胞作为染料加载细胞类型之间不会歧视之间测量细胞间隙连接通讯的变化。再次注射是选择的方法，但针刺针方法米格也HT工作，如果可以使用显微镜针对特定的细胞。 代谢合作测定是测量GJIC另一种方法。该方法是细胞间隙连接通讯24首生物测定标准之一。在该试验中，野生型中国仓鼠V79细胞（6-硫鸟嘌呤敏感）和6-硫鸟嘌呤抗性细胞在高密度，其中6-硫鸟嘌呤（6-TG）的抗性的细胞不代谢6-TG共培养于毒性代谢物，从而赋予阻力。共培养在毒性代谢物的通过间隙连接导致细胞死亡转让6-TG敏感的细胞的结果，其中的GJIC结果在6-TG抗性细胞的营救的抑制通过防止有毒的6-TG代谢物的转印从6-TG敏感的细胞。尽管该测定的一个优点是，它是微创，一个主要缺点是，它需要一个星期完成，这使得TOXI的广泛的剂量和时间依赖性的评估黑话以及机理研究等不切实际评估许多化学品。 对SL-DT测定，不利于机械的研究，需要通过不同的细胞类型来跟踪染料的迁移，其中显微注射是优选的方法。备选地，细胞可以用荧光染料的甲酯被预加载。染料的装载是水解亲脂性染料成其更亲水的形式，从而捕获在细胞中的色素细胞内的甲基酯酶的功能。这是预加载的荧光探针的较少侵入性的方式，而是以测量细胞间通讯，其中所有的单元都用这种方法预先加载的最常见的方法是在FRAP法11。 FRAP的优势又是评估单个细胞间隙连接的能力，这是极好的机理研究。然而FRAP的缺点在于:不能测量为毒理学评估细胞群，需要复杂的和非常昂贵的显微镜/激光器流式细胞仪，需要高度熟练的专业技术，是从由激光产生的自由基适度侵入性的，并且不利于高的吞吐量。 最近另一种技术已经开发;分子荧光探针（LAMP）方法25，这是基于新一代笼香豆素类荧光团的局部活动。类似于FRAP法，所有的细胞都预装了含有甲基酯染料，然而，这些染料成为通过用小剂量的紫外线，这可能产生显著降低ROS比FRAP激光器在随后的局部照明荧光。再次，该测定具有FRAP法的相同的限制。最近的其他测量细胞间隙连接通讯的进步是预加载和降落伞的技术，这也涉及使用甲酯荧光染料。预加载技术涉及暂停将细胞与荧光探针预装连同卸载同行然后被电镀并允许以形成汇合单层。染料的扩散然后可以用落射荧光显微镜26观察。在降落伞测定中，用荧光探针预装将细胞覆盖在卸载细胞单层，和染料的扩散用落射荧光显微镜27观察。再次，上述技术在技术上更具挑战性的高通量分析，并且必须在接近汇合立即镀和需要的细胞的再附着的时间滞后。 总体而言，对SL-DT测定是使用的设备，如CO 2细胞培养孵化器，生物安全柜和落射荧光显微镜，通常在大多数细胞培养的实验室发现一个相对便宜的，简单和通用的技术。以最小的经验和众多板块可以在一天，这有利于F为处理或筛选毒性以及用于预防或逆转毒物和毒素的作用的天然产物的化合物。此外，适应该测定用于高通量分析用机器人，利用自动荧光显微镜成像和分析系统将不得不筛选大量毒物和毒素的潜力。

该方法的总的目标是提供一种简单的，全面和相对廉价的技术来评估化合物的潜在毒性。这是一种体外方法，可以在多种细胞系中使用。配备有落射荧光显微镜标准细胞生物学实验室可以通过此法进行研究。 我们的细胞功能的基本知识已经高度依赖于在体外生物测定，并已成为医药品，环境污染物，以及食品生污染物的毒理学评估的必要成分。不幸的是，没有一个统一的体外生物测定系统，能够全面地满足所有毒理学评估的要求。许多体外测定的设计和优化，以评估以及评估一个特定的生物化学或分子端点。这些经常以高通量设置以反映一个扰动组合特定信号转导途径，如雌激素受体信号1。这一策略相当成功，但参与基因表达的信号转导通路的数量繁多，因此选择一个特定的信号通路，以评估相当复杂的任务。高通量的协议，目前正在开发的，并用于同时测量许多信号传导途径，这一直是一个方法克服一些单个分析的局限性。然而，并非所有的信号通路已经成功掺入更全面的办法，再加上新的信号传导途径正在不断地被发现，进一步复杂化这个评估过程。使用的体外方法广泛数字，特别是高通量系统中，综合毒理学评估也非常昂贵，并且不利于大多数单研究者领导的研究项目。 GJIC是一个过程TIG通过在电压变化，钙离子浓度，pH值，氧化还原平衡，由主要的细胞内信号转导途径和相互作用与膜和细胞骨架蛋白2,3-调节htly控制。因此，GJIC的抑制可以反映不同类型的细胞应激，不同的细胞功能的破坏，或不同的信号转导途径的扰动。克服利用有限的信号转导的生物测定的另一种方法是采取的生物现象优点，即许多，如果不是大多数，信号转导途径被进一步通过合作的细胞间的信号系统，通过间隙连接通道4-8调制。虽然间信号系统也很多，根据多路径控制，通过间隙连接通道间信号是最终被打开的通道的功能，部分封闭或完全封闭。这提供了可以使用各种可容易地测定的端点在体外生物分析系统7。考虑到组织的稳态设定点，需要畅通的渠道，确定化合物对间隙连接通讯的影响（GJIC）在确定化合物4,8的潜在的毒性作用更全面的方法。在本质上，在协调多个信号转导事件控制基因表达的核心作用这一关键的生物现象，使得毒性作用广泛评估。因此，评估GJIC生物测定是一个很好的起点，以评估化合物的潜在毒性。 用于评估GJIC最广泛的技术是基于预加载的细胞用荧光探针，然后从所装载的细胞或细胞相邻小区监控染料的迁移。技术预加载染料都涉及显微注射9，刮去探头11装载10和甲基酯</suP&gt;。手术刀装载荧光染料转移（SL-DT）的方法是刮负载的修改-染料埃尔Fouly 10开发的传输试验。而不是更微创刮，本报告的手术刀加载方法包括通过细胞减少侵入破坏（ 图1）单层圆刀片手术刀的温柔卷。这种技术的优点是细胞而非显微注射法的单细胞的群体的毒理学评估。此外，该测定的简单性允许在短时间内快速检测多块板而使用显微注射技术以及使用荧光探针的甲酯技术方法都是显著更费时，并且需要相当高的技术水平。 虽然没有单一的方法来满足研究细胞间隙连接通讯的所有需求;在SL-DT分析是一种简单的，相当便宜，多才多艺的分析，可以满足多种需求的各种化合物毒性的初步评估。主要优点包括:简单，设备或技能没有特殊需要所需要的其他方法，如微注射，荧光漂白恢复（FRAP）分析和分子荧光探针测定法，GJIC的在一个大一个快速和同时评估的局部激活后细胞的数目，有利于高通量设置用自动化荧光显微镜成像和分析，以及其适应性对于体内研究。

<table border="0" cellpadding="0" cellspacing="0" width="1238">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:312px;">WB-F344 rat liver epithelial cells</td>
			<td style="width:217px;">From Drs. J. W. Grisham and M. S. Tsao of the University of North Carolina (Chapel Hill, NC)</td>
			<td style="width:279px;">none</td>
			<td style="width:431px;">Provided by Drs. J. W. Grisham and M. S. Tsao University of North Carolina-Chapel Hill-NC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">35 mm Culture Plates</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">25 cm2 culture flasks</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">75 cm2 culture flasks</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-medium, an Eagles modified medium&#160;</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">Formula No. 78-5470EF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">fetal bovine serum</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">10437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% trypsin-EDTA&#160;</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">15050</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">phosphate buffered saline</td>
			<td style="width:217px;">homemade</td>
			<td style="width:279px;">see below for ingredient cat#&#39;s</td>
			<td>137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 2 mM KH2PO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">KCl</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3040-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NaCl</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3624-05</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3819--01</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3246-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lucifer Yellow CH, lithium salt</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">L0259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">rhodamine-dextran</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">R9379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1-methylanthracene</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">phenanthrene</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">P11409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">resveratrol</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">R5010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">D609</td>
			<td>Tocris Bioscience&#160;</td>
			<td style="width:279px;">1437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">acetonitril</td>
			<td>EMD</td>
			<td style="width:279px;">AX0145-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">37% solution formaldehyde</td>
			<td>JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">2106-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">#20 surgical blade</td>
			<td>Fine Science Tools Inc.&#160;</td>
			<td>10317-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">50 mL conical sterile tubes</td>
			<td>Thermo scientific&#160;</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon epifluorescence microscope&#160;</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">Eclipse TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon FITC dichroic cube</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">96107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;CCD camera&#160;</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">Nikon Cool Snap EZ CCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;imaging system.</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td>Nikon NIS-Elements F2.2 imaging system.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Image J</td>
			<td style="width:217px;">National Institute of Health</td>
			<td style="width:279px;">http://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
	</tbody>
</table>

gap junctional intercellular communication: a functional biomarker to assess adverse effects of toxicants and toxins, and health benefits of natural products

这个协议描述，其测量通过间隙连接通道间通讯，这是一个主要间过程，其中组织稳态保持手术刀放入荧光染料转移（SL-DT）技术。由毒物，毒素，药物等间隙连接通讯（GJIC）的中断已被链接到大量的不良健康影响。许多基于遗传人类疾病都与在间隙连接的基因突变。在SL-DT技术是GJIC在一个人口众多细胞的同时评估一个简单的功能测定。该测定通过细胞群体简要扰动细胞膜用手术刀刀片涉及预加载的细胞用荧光染料。然后荧光染料允许间隙连接通道横穿通过对相邻小区一段指定的时间。该试验然后通过加入福尔马林至细胞终止。在萤光灯的传播通过细胞群体rescent染料评估用落射荧光显微镜和图像被任何数量的形态软件包可用，包括在公共领域发现自由软件包进行分析。此法也适用于使用组织切片从处理过的动物各器官的体内研究。总体而言，对SL-DT测定可以起到广泛的体外药理学和毒理学的需要，并且可以潜在地适于使用自动荧光显微镜成像和分析的高通量设置系统在较短的时间以阐明更多的样品。

GJIC中断已被广泛用作生物标记物用于鉴定处于导致不利的健康影响14基因控制的nongenotoxic，后生水平的有毒化合物。例如，多环芳香烃（PAHs）是环境的无所不在的污染物，但在他们的后生毒性作为其分子结构15的函数而变化。较低分子量多环芳烃通常发现在相对较高的浓度比在许多不同的环境中的更高分子量的多环芳烃如被污染的河流沉积物到该香烟烟雾15,16。菲是一种低分子量PAH是具备三个苯环与被称为海湾地区的角度口袋里的一个例子。 图2是一系列具有递增剂量的菲的处理的WB-F344大鼠肝脏上皮细胞的SL-DT图像。请注意，在移民澳下降F中的荧光染料，荧光黄，用增加剂量。扫描的图像的区域可被确定为在控制（FOC）的一小部分。 图2的控制是细胞的溶剂处理的PAH溶于，即乙腈。通常SL-DT实验一式两份或一式三份进行用于与最少三个独立试验的每个试验性参数，这将是在这个实验中的剂量每次试验。然后将FOC值可被平均，统计分析，然后绘制（参见图3）。 类似的实验，也定期进行，以建立GJIC的毒物诱导的抑制，和之后的细胞转移到无毒药的介质从GJIC这种毒物诱导抑制恢复所需的时间周期的时间。 图4示出从抑制由菲两者的时间响应和恢复（苯丙氨酸）。对SL-DT测定也可用于确定潜在的机制由毒物和毒素dysregulate GJIC 4,16。这是通过预培养细胞在添加了dysregulates GJIC毒物或毒素的前一个选定信号传导途径的药物抑制剂进行。如果毒物或毒素不再dysregulates GJIC在于块信号传导途径，则此途径被确定为GJIC的调节途径，而这种途径可以排除如果毒物继续dysregulate GJIC所选药物的存在。 图5表明此主体，其中1-甲基蒽（1-MEA），另外三个环形的PAH抑制GJIC，但不是在D609的存在，这是磷脂酰胆碱特异性磷脂酶C（PC-PLC）的相当特异性抑制剂。因此，PC-PLC被裁定作为参与细胞间隙连接通讯的1-MEA-抑制作用的候选信号酶。 PC-PLC的作用已被证实为further实验4,16。总体而言，对SL-DT测定法是相当有利于开始参与毒物和毒素如何抑制（dysregulate）GJIC机制映射信令网络的过程。这种方法也可用于筛选天然产物，可以阻止从dysregulating GJIC一个毒物或毒素。例如，白藜芦醇，抗氧化剂在高浓度的红葡萄酒和花生制品发现可以防止1-MEA从抑制GJIC（ 图6）。 对SL-DT测定已适应组织切片从动物，特别是在肝脏17。全氟辛酸（PFOA）是一个8碳氟化脂肪酸是已知诱导肝癌12,17持久环境污染物。使用SL-DT技术PFOA显示抑制GJIC 在体外 WB-F344rat肝模型系统18。 体内随访研究DETermined该PFOA也抑制GJIC与PFOA治疗F344大鼠的肝脏中，从而证实在体外模型系统12,17。 图7是从该后续实验的荧光图像示出与车辆和PFOA处理的大鼠的肝组织的染料扩散。 <img alt="图1" src="/files/ftp_upload/54281/54281fig1.jpg" /> 图1:手术刀染料加载步骤的卡通形象。示出手术刀装载过程涉及轧制叶片而不是通过细胞水平切片以最小化细胞损伤的图。 <img alt="图2" src="/files/ftp_upload/54281/54281fig2.jpg" /> 图2:染料的代表性荧光显微图像通过间隙连接传播。 WB-F344大鼠肝脏上皮细胞进行处理15分钟，在压痕剂量菲，一个普遍的PAH环境中发现的。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54281/54281fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54281/54281fig3.jpg" /> 图3:菲处理的WB-F344大鼠肝脏上皮细胞的剂量响应曲线。 SL-DT图像的区域被计算，然后记录为控制，这是与车辆（乙腈）处理的细胞的一小部分。每个数据点是三次独立实验的平均值和误差棒是在95％的置信限为15分钟的曝光时间后的各剂量的标准偏差。一个四参数逻辑函数来拟合通过数据点的线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54281/54281fig3large.jpg" target="_blank">请cli来</a>这里完蛋查看此图的放大版本。 <img alt="图4" src="/files/ftp_upload/54281/54281fig4.jpg" /> 图4: 菲处理的WB-F344大鼠肝脏上皮细胞的时间和时间恢复响应曲线。曝光后的10分钟GJIC的完全抑制之后，含介质菲丙氨酸（Phe）被切换到Phe-免介质中，然后将细胞从抑制恢复监测。 SL-DT图像的区域被计算，然后记录为控制，这是与车辆（乙腈）处理的细胞的一小部分。每个数据点是三次独立实验的平均值和误差棒是在95％置信限的标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54281/54281fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> her.within页="1"&gt; <img alt="图5" src="/files/ftp_upload/54281/54281fig5.jpg" /> 图5:GJIC 抑制由1-甲基蒽依赖于磷脂酰胆碱特异性磷脂酶C. 1-甲基蒽（1-MEA）是在环境中发现的一种普遍的PAH。左侧面板代表与车辆处理的细胞（乙腈，0.35％体积/体积）。中间面板代表15分钟用70μM1-MEA处理WB-F344大鼠肝脏上皮细胞。右侧面板代表第一预处理20分钟用50μMD609，磷脂酰胆碱特异性磷脂酶C抑制剂的细胞，然后加入70μM1-MEA的另外的15分钟。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54281/54281fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 281fig6.jpg"/&gt; 图6:白藜芦醇抑制细胞间隙连接通讯的抑制1-甲基蒽。左侧面板代表与车辆处理的细胞（乙腈，0.35％体积/体积）。白藜芦醇是红酒和花生制品中的抗氧化剂。中间面板代表15分钟用70μM1-MEA处理的细胞。右侧面板表示第一预处理20分钟以50μM的白藜芦醇，接着加入70μM1-MEA的另外的15分钟的细胞。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54281/54281fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54281/54281fig7.jpg" /> 图 7: 雄性F-344大鼠肝切片细胞间隙连接通讯的 体外 SL-DT测定。左侧面板代表肝脏做卷烟中分得一杯羹UE从与车辆控制，二甲基亚砜，处理24小时大鼠。右面板代表了肝组织从100毫克腹腔给药后大鼠切片后24小时/千克全氟辛酸（PFOA）。 PFOA，一个普遍的环境污染物，是一种肿瘤促进过氧化物酶体增殖诱导肝肿大和证实到方框使用体外模型间隙连接。切口加载染料从车辆和PFOA处理的动物肝切片，然后将其固定，修整并切片进行。从环境与健康展望 12再现。比例尺= 20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54281/54281fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products 

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

本协议描述，通过间隙连接通道测量细胞间通讯手术刀装载荧光染料转移技术。间隙连接通讯是一个主要的细胞过程，其中组织稳态维持这种细胞信号中断了不良的健康影响。

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

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Nanyang Technological University

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JoVE Journal

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Cell Biology

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Unit 1

Cell-Cell Interactions

SCIENTISTS IN ACTION

14.9K 次观看.  Michigan State University. 本协议描述，通过间隙连接通道测量细胞间通讯手术刀装载荧光染料转移技术。间隙连接通讯是一个主要的细胞过程，其中组织稳态维持这种细胞信号中断了不良的健康影响。 

视频: Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

科研

生物学

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.
Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.
In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.
We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.

使用不固定，冷冻的组织，以研究自然的粘蛋白分布

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

一种碘-黄色荧光荧光蛋白-缝隙--细胞间通信方法

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

风险蝴蝶捕获繁殖计划，以提高生命历史知识和有效的异地保护技术

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

使用便携式 qPCR 系统对 马特里卡里亚查莫米拉进行现场识别

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

在靶标和标记下使用裂解分析植物中的H3K4me3修饰

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...