我们感谢 Dipa Bhaumik, 艾伦. 米勒和鲍勃. 休斯的技术援助。CGC 提供的菌株是由 NIH 研究基础设施项目办公室 (P40 OD010440) 资助的。我们感谢格鲁吉亚伍兹和 Lithgow、Kapahi 和 Melov 实验室的成员进行有益的讨论。M.L. 得到国家卫生研究院 (NIH) 培训补助金 T32 AG000266 和埃里森医学基金会/美国老龄研究联合会博士后奖学金的支持。这项工作得到了来自 BioAge 实验室、拉里 l. Hillblom 基金会赠款以及国家卫生研究所赠款 UL1024917、支持 Geroscience 跨学科研究联合会的赠款和 1R01AG029631-01A1 G.J.L。

1. 准备缓冲区
<ol><li>
LB 汤
	<ol>
<li>使用预混合颗粒 (请参阅材料表) 并按照制造商的协议进行准备。</li>
	</ol>
</li>
	<li>
线虫生长培养基 (NGM) 琼脂
	<ol>
<li>混合23克琼脂, 3 克氯化钠, 2.5 克 bacto 蛋白胨, 和去离子水 0.972 l 高压釜和让冷却到60°c, 然后添加25毫升的1米磷酸钾 (KPO4) 缓冲 (pH 6.0), 1 毫升的1米硫酸镁 (MgSO4), 1 毫升1米氯化钙 (CaCl2), 1 毫升5毫克/毫升胆固醇 (乙醇)。</li>
	</ol>
</li>
	<li>
次氯酸盐溶液
	<ol>
<li>混合5毫升5米的 KOH, 4 毫升次氯酸钠溶液 (6%), 和去离子 H2O 的总体积为50毫升。</li>
	</ol>
</li>
	<li>
S 基
	<ol>
<li>混合 5 g 的氯化钠, 1克的 K2HPO4, 6 克的, g 的 2 PO 4, 和脱电离水到1升, 然后蒸压釜。</li>
	</ol>
</li>
</ol>2. 浓缩的大肠杆菌的制备 (OP50)
注: 此步骤将1升的饱和大肠杆菌溶液浓缩为25毫升的 S 基缓冲液。
<ol><li>接种5毫升蒸压 LB 汤 (步骤 1.1) 与单一的大肠杆菌菌落 (应变 OP50), 并孵化4-6 小时在37°c 与震动 (250 rpm)。使用0.1 毫升的这个解决方案, 接种1升的磅在2升锥形瓶。1升烧瓶过夜 (12-16 小时) 在37°c 与震动 (250 rpm) 孵化。</li>
	<li>离心机 1 L 隔夜文化在500毫升离心机瓶为5分钟在 1万 x g 和4°c 给细菌和醒酒离开剩余的媒介。并用重悬颗粒在25毫升的 S 基部 (步骤 1.4)。存储在4摄氏度。</li>
</ol>3. NGM 培养板的制备
<ol><li>对于用于产生大量蠕虫的大众培养板, 将30毫升的熔融 (60 °c) NGM 琼脂 (步骤 1.2) 放在层流柜中, 用自动液体配药装置或血清吸管在10厘米培养板上。允许干燥过夜。</li>
	<li>吸管2毫升的浓缩 OP50 每10厘米板, 蔓延到完全覆盖琼脂表面通过倾斜和旋转板, 然后允许板材干燥。贮存板在摄氏4摄氏度, 长达2周。</li>
	<li>对于用于筛选的高通量检测培养板, 利用自动8通道分配器将0.15 毫升的熔融 NGM 琼脂分配到层流柜中96个井板的每个井中。使用小心, 以确保井没有气泡, 因为这些将使洞穴的蠕虫, 将损害的化验在那井。允许干燥过夜。贮存板在摄氏4摄氏度, 长达2周。</li>
</ol>4. 温度敏感的制备 (ts) 无菌的c 线虫大众文化
<ol><li>要开始文化, 使用 "蠕虫采摘" (一个玻璃吸管与附有白金导线或制造的蠕虫挑选) 在解剖显微镜下, 转移20个蛋 (应变 TJ1060: spe-9 (hc88) I; rrf 3 (b26) II) 上的质量培养板 (s) 在步骤3.1 中准备。然后, 孵化板在20°c 6 天 (允许超过一个世代的增长, 因为你的目标是收集的后裔20鸡蛋移动)。</li>
	<li>用解剖显微镜观察妊娠成人 (在子宫中存在的卵) 的存在。用玻璃吸管将2-3 毫升的 S 基在盘子上, 收集妊娠成虫。用手将液体旋转在盘子周围, 然后用玻璃吸管将液体收集到15毫升管中。</li>
	<li>向下旋转管 (1000 x g 1.5 分钟20°c) 到颗粒蠕虫, 然后吸出上清。将10毫升次氯酸盐溶液 (步骤 1.3) 添加到蠕虫颗粒中, 用手快速摇动管子, 在 5 s 的顶端到底部方向. 孵育5分钟, 每2.5 分钟晃动一次。</li>
	<li>再次, 向下旋转管 (1000 x g 为1.5 分钟);颗粒应为淡黄褐色。从上清中吸取。将10毫升次氯酸盐溶液加入卵团, 并大力摇动管。孵育1-3 分钟, 偶尔晃动, 并通过解剖显微镜监测外观。目视确认只剩下卵子, 然后继续下一步。</li>
	<li>向下旋转管 (1000 x g 1.5 分钟) 并吸取上清。颗粒应该是白色的, 没有褐色的着色。 注: 在去除次氯酸盐溶液后, 使用无菌技术和缓冲器是至关重要的, 因为污染 (特别是细菌和真菌) 会破坏实验, 浪费宝贵的时间和试剂。本研究使用了过滤层流柜和移动液体只使用无菌吸管和提示。</li>
	<li>在无菌空间打开管子。用 S 基部清洗颗粒3次, 向下旋转 (1000 x 克1.5 分钟), 每次吸走上清液。在2-3 毫升的基础上重新悬浮卵颗粒。这些卵现在可以用于一个夜间舱口在缓冲收集 L1 幼虫 (步骤 5)。</li>
</ol>5. 在缓冲中孵化c 线虫卵以获得同步培养基 (制备 L1 幼虫c 线虫)
<ol><li>醒酒的鸡蛋颗粒和缓冲成一个无菌的玻璃培养皿与封面。用2-3 毫升 S 基的血清学吸管将剩余的卵从管子中冲洗成一道菜。添加5毫升的 S 基部, 以缓解拥挤, 因为这可能会鼓励孵化, 如将轻摇晃 (20 rpm 与肚皮舞者轨道振动筛)。孵化隔夜在20°c, 允许蛋时间孵化 (16-24 小时)。由于没有食物, 所有的幼仔将在 1st幼虫阶段 (L1) 中被捕。 注意: 轶事证据表明, 新鲜的 L1 制剂 (16-36 小时后的卵子收集) 产生最同步的成虫种群。</li>
	<li>用血清学吸管在15毫升管中收集 L1s。在离心机上旋转 L1s (1000 x g 1.5 分钟), 吸走上清, 然后重复, 直到所有的 L1s 从孵化盘中收集, 同时留下10毫升的 S 基为下一步。</li>
	<li>从含有 L1s 的管中, 微出0.01 毫升 (混合后立即), 并将其分配到非种子选手 (无大肠杆菌) NGM 板上. 计算盘子上的 L1s 数。重复10次并获得每个整除中的 L1s 数的平均值。一般情况下, 预计每µL 1-3 L1s (当开始与1质量培养板包含20个蛋)。</li>
	<li>用血清吸管测定 L1s 的 S 基体积。然后用确定的平均每0.01 毫升蠕虫数量推断出, 在上一步中得到的 L1s 总数。一般情况下, 预期在 1000万 L1s (每块含有20鸡蛋的质量培养板)。</li>
	<li>要使用这种文化在96井化验, 稀释 L1 悬浮到 1 L1 每µL 在一个无菌的圆形培养基瓶, 然后进行步骤7。如果此区域性用于生成其他C. 线虫大众区域性, 则继续执行步骤6。</li>
</ol>6. 大型同步培养制剂
注意: L1 解决方案可用于快速增加蠕虫种群。
<ol><li>要生成大的蠕虫数字与 L1 解决方案, 分配多达 5000 L1s 每一个期望数量的大众文化板块。收集妊娠成人2.5 天后 (20 °c), 如步骤 4.1-2 所述, 并着手步骤4.3 收集鸡蛋。</li>
	<li>正如人们所说的, 反复的次氯酸盐收集可以强调被征服的人口, 在将剩余的人口置于次氯酸盐治疗之前, 通过采摘卵子来分裂种群, 从而使多个世代的人口不反复地接受次氯酸盐溶液治疗。</li>
</ol>7. 96 井检测板的设置
<ol><li>允许化验盘 (在步骤3.3 中准备) 达到室温。然后, 在层流流柜中, 添加5µL 的浓缩 OP50 (在步骤2.2 中准备) 给每个井。与以前一样, 使用自动8通道分配器。</li>
	<li>使用自动8通道分配器在每个井中添加10µL 的 L1 悬浮液。这将产生平均10蠕虫每井, 因为它们存在于悬浮在 1 L1 每µL。为促进 L1s 从浆料中均匀分布, 将 L1s 从一个圆形介质瓶中, 与一个适度缓慢转动的搅拌棒混合在一起。</li>
	<li>盖板, 盒批板, 孵化25°c 2 天。</li>
</ol>8. 治疗 1 st天成人C. 线虫与化学品
<ol><li>从冰箱中取出化学库板, 在继续操作前允许板材达到室温。 注: 这里所描述的化学库板块是每个井中含有离散化合物的96个井板, 所有这些都处于相同浓度, 并在亚砜中溶解。这些库不使用外列, 因此每个板块最多有80种不同的化学物质。在这里描述的协议中, 库集中度为10毫米。考虑溶剂对表型的影响是很重要的。虽然这项研究在亚甲基亚砜浓度为 0.5%, 但这一水平从来没有超过这些寿命化验和低吞吐量化验, 其中更有可能选择一个复合库存溶液浓度。此协议不超过 0.25%, 这是一个级别, 在这种情况下, 寿命修改不会发生16, 至少在 N2 应变中。</li>
	<li>在使用前, 在微板块振动筛上轻轻摇动 60 rpm 的盘子, 1 分钟即可。</li>
	<li>使用自动96井液体处理程序, 将0.75 µL 的化合物 (或亚砜) 从库板转移到检测板上。液体输送过程中的吸管深度被仔细控制, 这样液体处理者的吸管尖端将0.75 µL 的体积传递到琼脂表面的15µL 液体中 (与蠕虫和浓缩 OP50 从步骤7。1-2), 不刺穿在井中的琼脂表面。不要使用手动8或12多通道吸管, 除非在非常小的屏幕 (< 10 板), 因为它们可以增加错误, 往往导致穿刺的琼脂表面, 这危及化验。</li>
	<li>用亚砜 (二甲基亚砜) 制备化学库负控板, 假设所测试的库采用亚砜作为溶剂。为每5个测试板制作1个负控板, 最多10个负控板。如果可能的话, 也做一个正面控制板 (NP11在20毫米, 为最后的测定浓度0.1 毫米, 是有效的为此目的; 请参见 "代表性结果" 部分)。不要将化学品 (测试或控制) 添加到板块的2外部柱上, 因为它们特别容易脱水。</li>
</ol>9. 干燥文化
<ol><li>从琼脂表面除去液体, 在层流柜中干板4-8 小时。这是至关重要的, 这些板块是完全干燥, 但不会变得干燥。由于有些盘子的干燥时间比其他板块要长, 所以要密切监视所有的盘子, 并在干燥后立即清除。通过仔细目视检查板材的各个井, 确认干燥。 注: 根据包括机柜排气速率的因素, 必须以经验的确定时间。这个干燥步骤可能需要大约4-8 小时的一个完整的橱柜。</li>
	<li>用透明薄膜密封板, 然后放在板盖上。</li>
	<li>四十五年代在 1000 x g 旋转96井化验盘。</li>
	<li>商店板块倒在25摄氏度。</li>
</ol>10. 为长寿而评分的c 线虫文化
<ol><li>监视阴性控制板直到 > 95% 死亡被观察。检查每周的前两周和每天的车牌。相同应变和物种的秀丽群可以通过试用17显示显著不同的寿命。 注: 在这里描述的寿命测定, 与 TJ1060 动物养殖25摄氏度, 95% 的死亡率, 这些人口发生了从成年日16-20。</li>
	<li>当负控井被认为达到 > 95% 标记时, 在桌顶离心机上自旋下所有96井检测板, 微板块适应转子 (250 x g 为四十五年代在20°c)。</li>
	<li>把控制板上的井都分了。这是通过计数和记录在每个井活和死的动物的数量完成。死蠕虫可以被区分从活蠕虫由他们的完全缺乏运动。通过将96井板从大约7厘米滴到解剖显微镜表面, 同时通过目镜进行任何诱发反应, 激发活蠕虫的运动。死蠕虫不会反应, 附近的实验室伴侣可能不欣赏重复的噪音。</li>
	<li>对于测试板, 计数和记录只含有活蠕虫的水井。当观察到活蠕虫时, 把所有活着的和死的动物都算在井里, 连同井的位置和盘子。 注: 在 > 99% 的阴性控制蠕虫死亡后, 重新评分盘子通常是有用的。在大规模的屏幕上, 这可能是最好的点做初始评分。在这两种情况下, 评分/重新评分如上文所述</li>
</ol>11. 重新测试化合物
<ol><li>在仅测试少量候选化合物 (< 320 化合物) 时, 与重新测试或候选屏幕 (见代表性结果) 一样, 限制对32中心井的化验, 以尽量减少可能的板块效应。这使得2口最接近板块边缘的油井未经处理, 但仍含有琼脂、食物和蠕虫。</li>
</ol>

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J., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+dietary+restriction+for+genetic+epistasis+analysis+and+gene+discovery+in+C.+elegans.">Optimizing dietary restriction for genetic epistasis analysis and gene discovery in C. elegans.</a> PLoS One. 4 (2), 4535 (2009).</li><li>Hamilton, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+for+longevity+genes+in+C.+elegans.">A systematic RNAi screen for longevity genes in C. elegans.</a> Genes Dev. 19 (13), 1544-1555 (2005).</li><li>Frankowski, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide+and+dimethyl+formamide+increase+lifespan+of+C.+elegans+in+liquid.">Dimethyl sulfoxide and dimethyl formamide increase lifespan of C. elegans in liquid.</a> Mech Ageing Dev. 134 (3-4), 69-78 (2013).</li><li>Lucanic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+genetic+background+and+experimental+reproducibility+on+identifying+chemical+compounds+with+robust+longevity+effects.">Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects.</a> Nat Commun. 8, 14256 (2017).</li></ol>

提交人声明, 任何支持这项工作的资助实体都没有对数据收集、结果分析或发布决定作出任何投入。

在这里, 我们描述了一个简单的方法来培养的C. 线虫在96井板块。我们描述这些文化用于化学筛选和寿命测定, 但它们可以用于多种类型的化验。虽然以前已报告了化学筛选的多井养殖条件8, 包括用于寿命分析的10, 但此处描述的检测方法在几种方面有所不同。这里描述的寿命试验利用96井板, 依靠无菌突变体 (而不是化学杀菌), 使用琼脂培养条件, 使用简单的液体处理来移动蠕虫, 并在终点手动得分。这种方法中使用的不同方法可能有助于研究人员寻找包括这些条件的筛选法。
该协议的关键要素主要集中在为检测而制作的板材的质量上。首先, 在制作盘子的时候, 琼脂表面是没有气泡和其他缺陷的。这些变化在琼脂表面几乎总是导致挖洞和损失的井。第二, 该协议依赖于干燥在安装和下药阶段储存的液体。这是至关重要的, 这些液体被完全去除, 但琼脂不会变得干燥。有趣的是, 在水井中没有完全干燥的蠕虫 (在液体中游泳) 比正常的干井活的时间长, 而过度干燥的盘子会导致琼脂的脱水和开裂, 导致蠕虫的挖洞和损失。该议定书的另一个关键内容是保持无菌条件。与任何寿命检测一样, 有很多工作涉及的板块设置, 并有许多机会和时间来破坏污染, 以解决和增长的板块。
虽然此处描述的寿命分析对于筛选线虫中的化合物非常有用, 但它仅限于特定的遗传背景, 因为它依赖于温度敏感 (ts) 无菌菌株;我们使用 TJ1060, 它有高不孕显性。这限制了可用于筛查这种方法的菌株。其他方法, 包括将ts无菌突变转化为所需的背景或使用化学杀菌。我们没有尝试化学杀菌的这种化验, 但它应该是可能的。潜在的障碍包括将灭菌器放在正确的阶段和适当剂量的蠕虫上。此处描述的协议使用的液体分配器是快速的, 但似乎没有有效地免除成年人。因此, 这些蠕虫发展的板块, 因此必须处理与灭菌化学品的板块。确定最佳剂量和时机对成功至关重要。替代策略包括使用一种可以移动成年人的配药方法。过去, 我们发现, 能够识别和排序成年人的机器通常比简单的液体处理器慢得多, 这可以免除悬浮幼虫的均匀浆料。
一般而言, 我们使用这种方法快速筛选化学药品和治疗, 以对寿命产生巨大的积极影响。当使用多剂量、高复制数和密切监视始终保持和散置在测试板之间的负控制时, 该方法特别有效。在设计和实现本手稿中描述的屏幕时, 积极的控制也很有用。然而, 要成为一个有效的积极的, 控制将需要相当强大和/或高度健壮。当屏幕最初被部署时, 无法识别出一个适当有力和健壮的化合物。目前, 有许多报告在 "老龄化" 的文学可能的候选者的积极控制有用的屏幕上描述。如本手稿的 "预期结果" 部分的 "候选屏幕" 部分所述, 在图 2和表 1中, 复合 NP1 是此屏幕的有效正控件。我们通常确认从这些屏幕上的正面命中与标准寿命测定3毫米培养板。

这里描述了一种用于对秀丽线虫寿命产生影响的化合物高通量筛选的协议。该协议本身很容易适应其他表型屏幕, 包括使用记者C. 线虫菌株的研究。该协议成功地用于识别一种新的生物活性化合物, NP1 (硝基苯哌嗪 1), 通过饮食限制机制, 强烈延长了线虫的寿命。NP1 及其对寿命的影响, 包括对游戏中的遗传通路的描述, 以前在别处被描述为1。该报告还包括对屏幕的大规模实现所产生的结果的描述。在这里, 我们更详细地描述了屏幕本身的方法和设置, 这对于小规模和大型应用程序都是可伸缩的。
导致创建此协议的目标是开发一个"线虫"文化平台, 允许对大型化学图书馆进行快速筛选, 以用于新的生物活性化合物。虽然在本协议开发之前已经执行了化学屏幕, 但它们主要是小规模和/或候选类型方法2,3。事实上, 实验室中的大多数屏幕上都使用了抗应力测试方法, 其中的命中被筛选为长寿效应4。这项研究只是寻求直接筛查寿命效应。值得注意的是, 在使用线虫进行大规模化学筛的时候, 科学文献中很少有描述。事实上, 与其他类型的屏幕5,6,7, 大规模化学检查使用C. 线虫似乎是在就业不足。
在C. 线虫中有两种大型化学筛的描述, 用作开发这种方法的基础。第一个是来自彼得罗伊实验室的一项优雅的研究, 它使用化学筛来识别能扰乱动物发育的化合物。随后, 该研究追踪了一个前诱变筛, 以确定这些化学物质的基因指标8。该项研究中所述的协议使用了24井酱, 这对于该研究的具体需要来说是行不通的 (图书馆的化学物质有限, 可用的孵化器空间有限)。另一项研究是迈克尔 Petrascheck 的创新和雄心勃勃的小分子筛寿命延长化学品9,10。那项研究使用了384井板材和液体文化。这个屏幕的另一个主要特点是使用 FUDR (5-fluorodeoxyuridine) 化学抑制后代生产。在对该项研究中所述的议定书进行了大量审议之后, 避免了化学杀菌和线虫的液体培养。
尽管 FUDR 被广泛应用于线虫寿命实验, 但人们担心 FUDR 可能导致意外的药物相互作用, 或者 FUDR 本身可能对寿命有一定影响。最近, 后者的关注已得到验证, 至少在某些浓度, 特别是在25°c11。为了规避子代污染的问题, 采用了线虫应变 TJ1060, 该菌株具有两种独立的温度敏感突变 (spe-9 (hc88) I; rrf-3 (b26) II), 它可以消除精子的生产并证明是有用的。对于同步种群的大众文化,12。这种菌株在25摄氏度时完全不育, 但在15-20 摄氏度培养时会产生后代。这项研究使用传统的文化条件, 蠕虫爬行在琼脂表面, 因为获得的结果与液体培养不总是重现与传统的琼脂板块。虽然这一现象尚未完全理解, 但已经证明, 在液体中培养的蠕虫以不同的方式响应 DR (饮食限制), 而不是在标准媒体13、14中培养的蠕虫。因此, 液体媒体可能会掩盖一些 mimetics 博士, 否则会被屏幕识别。
通过对琼脂培养条件的研究, 探讨了在本试验中对油井性能进行评分的不同选择。虽然有各种自动化或影像学的分析方法, 但它们需要获得和部署技术上具有挑战性的设备, 其中大部分费用高昂。在考虑这些选项的同时, 必须提到所有类型的以前的生命周期屏幕。最终, 这项研究使用了一个评分方法, 适应了小西尔维娅李实验室的整个基因组干扰屏幕的寿命表型15。具体地说, 所有的寿命板都是以同样的方式建立的, 但只有负控制被监测。当阴性控制板被证实有接近完全死亡, 测试板材由手被得分。在这个大规模的屏幕, 积极的确认通过重新测试与新鲜准备的化学品, 使用三次重复的主要阳性。

<table border="0" cellpadding="0" cellspacing="0" style="width:1311px;" width="1311">
	<colgroup>
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	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">LB Broth Miller Granules</td>
			<td style="width:363px;">VWR</td>
			<td style="width:344px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Unispense&#160;</td>
			<td>Wheaton Science Products</td>
			<td></td>
			<td>automated fluid dispensing apparatus</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">96 well assay plate; COSTAR 3370</td>
			<td style="width:363px;">Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multidrop 384; automated 8 channel dispenser</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">manufactored worm pick</td>
			<td style="width:363px;">Genesee Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Belly Dancer Orbital Shaker</td>
			<td>Stovall Life Science inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">microplate shaker; MTS 2/4 Digital</td>
			<td>IKA Works, inc.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">automated 96-well liquid handler; Biomek FX Liquid Handler</td>
			<td>&#160;Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">automated 96-well liquid handler; Bench Top Pipettor&#160;</td>
			<td>Sorenson Bioscience, inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">manual 8 or 12 multi-channel pipettes</td>
			<td style="width:363px;">we use rainin&#8230;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">transparent 96 well film cover; TempPlate RT Optical Film&#160;</td>
			<td>USA Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">table top centrifuge; Centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple method for high throughput chemical screening in caenorhabditis elegans

秀丽线虫是测试生理化学效应的有用有机体。整个有机体小分子筛为识别生物活性化学结构提供了显著的优势, 可以修改复杂的表型, 如寿命。这里描述的是一个简单的协议, 用于生产数以百计的96井文化板块, 每个井中有相当一致的数目的C. 线虫。接下来, 我们指定了如何使用这些区域性来筛选数以千计的化学物质, 以影响线虫线虫的寿命. 该协议使用温度敏感的无菌菌株, 琼脂板条件, 简单的动物处理, 以促进快速和高通量生产的同步动物文化进行筛选。

我们首先从使用此协议的96井寿命检测的代表性结果进行检查。在第一个示例中, 该方法用于筛选3万种化学物质, 以成功地识别延长线虫的寿命的化学物质,并包括后续重新测试屏幕的最佳性能化学品使用的结果相同的协议。这些结果以前被描述为1。第二个示例在候选化合物的较小规模屏幕中使用相同的协议。
大规模屏幕和重新测试:大规模的屏幕测试3万化合物是以单一剂量, 重复进行。因此, 每种化学物质都在两个水井中试验, 预计总的动物种群为20。为了完成这一目标, 在3月的时间内筛选出了三组1万化合物 (重复), 以覆盖整个3万种化合物。这是在一定程度上完成的, 以确保在化验结束时可控量的人工评分, 并要求在每套260的96井化验板。在图书馆存量板块中, 只有80口井含有复合物;一次复制1万个化合物做125个化验盘。本研究使用2复制与10负控制板在每套。为了提高吞吐量, 在评估负控制人群经历了99% 的死亡率时, 板块被评分。所有测试井然后被检查为活蠕虫。
有一只活蠕虫的水井中的化学物质被称为命中。含有1多种蠕虫的油井中的化学物质也被称为命中, 另外, 这些井中的所有蠕虫 (活的和死的) 都被计算成一个简单的分数 (活的分数的百分比, 活蠕虫的数量除以死蠕虫的数量)。因此, 库中的每种化学物质都可以被称为 "命中两次", 而这些命中可能, 但可能不会, 有与之相关的分数。512种不同的化学物质被称为命中 (至少一次) 从3万化合物的屏幕。该屏幕的目的是确定有效和/或健壮 (可重现) 亲长寿的效果。这两种不同的属性可能具有不同的效果。有效的化学物质可以显著地增加寿命, 在这种情况下, 我们会期望高比例的蠕虫活在该化学品的良好。对于强健的化学物质, 两种复制都有望有活蠕虫。命中名单根据这些标准排列。179的化学物质产生了很高的活度分数, 或者在两种复制中被击中, 因此选择重新测试。大多数剩余的化学物质被称为命中, 但没有选择重新测试, 只包含一个在复制井中活着的蠕虫。
对命中进行重新测试, 与主屏幕类似, 除非有3种复制 (预期总的动物数量为 30), 并且仔细地定量测定未经处理的控制板中动物的寿命。这些变化增加, 以帮助比较测试和控制井。179的化学命中被重新排序, 稀释到10毫米, 并移动到96井板。只有内部24口井被用于重新测试化验 (本议定书的目前版本将使用32井, 如协议部分中所述的重新测试屏幕), 这些化合物被重新测试了三个与六阴性对照试验板用亚砜溶剂处理。一个负控制板定期得分以获得总生存期 (图 1A)。当大约95% 控制动物死了, 所有板材完全地被得分了 (所有被处理的活和死的蠕虫计数了)。
要从重新测试结果中调用命中, 在负控制 +2 标准偏差 (SD) 的平均活度分数上进行了截割。对于测试井, 平均活度 (在评分时) 分数计算通过平均活分数的百分比从三复制为每个井。对于这一特定的检测, 负控制井的平均活度分数计算为平均三复制48井 (2 套三个负控板)。用于从重新测试化学集调用命中的 sd 是48负控制平均活分数的 sd。使用此策略, 本研究发现179种化合物的重新测试库中有57次命中, 表明总共有32% 的重新测试化学物质测试为阳性 (图 1B)。负控制和试验井平均活分数的 Binning 表明, 测试井优于控制井 (图 1C)。
候选屏幕:小规模的屏幕可以执行类似于上面所述的重新测试的方式。在这里, 我们描述的结果, 从一个候选屏幕与139化合物。这些候选者被组装成可能促进长寿的结构。对于这个屏幕, 我们在两种不同剂量 (50 µM 和100µM) 上进行了5次测试板的复制。此外, 我们使用了8负控制板, 单阳性控制 (NP1) 板含有两种不同的剂量;50µM 和100µM。
在90% 的动物死亡之前, 对阴性对照板进行了监测。当时, 在所有控制井中计数活的和死的蠕虫, 以及含有活蠕虫的测试井, 都能获得油井的分数。得分时活着的百分比被用来计算每个井平均活分数的百分比。对于负控制, 32 口井有8个复制的活分数%。对于阳性对照组, 每个剂量的4次复制平均有4个好分数。命中被要求为井以平均百分比活比分比平均阴性控制百分比活比分 +2 SD。这一截距从候选屏幕上留下了14次命中, 排除了所有的负控制井。该截止还包括所有的阳性对照井治疗100µM 和50% 的阳性对照治疗50µM。这里给出了这些结果, 因为作废平均活分数为50µM (图 2A) 和100µM (图 2B) 测试屏幕。
最后, 所有板块在一个较晚的时间点重新得分, 这对应于一个时间, 当超过99% 的负控制治疗动物死亡。这些结果表明, 正控制井远未执行负控制和测试井 (图 2C)。而测试井比负控制略好。后者的结果是有偏见的迹象表明, 许多测试井似乎表现出的毒性相对于控制治疗 (表 1), 从而混淆这一简单的解释。这是预期的, 因为候选图书馆是一个真正的屏幕上的化学品与未知的生物活动在C. 线虫, 而重新测试屏幕基本上是预先筛选的化合物, 不是有毒的。由于这一主要的屏幕是为延长寿命的化合物, 显着有毒化学品不应该在重新测试集。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56892/56892fig1.jpg"> 图 1: 高吞吐量屏幕和重新测试的代表性结果 (A) 在重新测试检测中使用的具有代表性的负控制板的生存曲线。这条曲线是在24井中使用的所有活和死蠕虫的评分在18天的试验中所用的4次。在成年18天, 对控制进行了评估, 有5% 生存和所有控制试验和板块, 然后也得分。(B) 表, 汇总屏幕结果并重新测试。(C) Binning 从重新测试屏幕的控制和测试条件中平均活分数的百分比。对于控制, 分数是从48口井, 每一个组成的平均3复制。测试结果来自179口井, 每一个由三个复制组成的平均分数。图 1从以前的出版物1中复制。<a href="//cloudflare.jove.com/files/ftp_upload/56892/56892fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56892/56892fig2.jpg"> 图 2: 候选屏幕的代表结果 (A) Binning 50 µM 候选屏幕的结果。139试井结果代表了5个复制中平均活分数的百分比。32负控制结果由8个复制的平均活度分数组成。4阳性控制结果包括平均活分数从4复制 (16 个总井包含 NP1 在50µM)。(B) Binning 100 µM 候选屏幕的结果。所有与 (A) 相同, 只是测试井和阳性对照治疗的µM 浓度为100。同样的负控制板也显示在两个 (A和B) 中。(C) 代表的结果是在相对极端的较晚时间点重新评分板块。如果只是在这个时间点上计算出有活蠕虫的油井百分比, 这对测试井有偏见, 如主文本和表 1中所述, 积极的控制会显著优于所有其他人。<a href="//cloudflare.jove.com/files/ftp_upload/56892/56892fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td colspan="6">平均活分数的 Binning</td>
		</tr>
<tr>
<td>分组评分</td>
			<td>全部死亡 (0)</td>
			<td>1-10</td>
			<td>11-20</td>
			<td>21-30</td>
			<td>31-40</td>
			<td>> 41</td>
		</tr>
<tr>
<td>重新测试</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td># (-) 控制</td>
			<td>16</td>
			<td>31</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
<tr>
<td>测试井 (50µM)</td>
			<td>46</td>
			<td>69</td>
			<td>50</td>
			<td>8</td>
			<td>3</td>
			<td>3</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>候选屏幕</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td># (-) 控制</td>
			<td>0</td>
			<td>11</td>
			<td>21</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
<tr>
<td># (+) 控制 (50µM)</td>
			<td>0</td>
			<td>0</td>
			<td>2</td>
			<td>2</td>
			<td>0</td>
			<td>0</td>
		</tr>
<tr>
<td># (+) 控制 (100µM)</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>2</td>
			<td>2</td>
		</tr>
<tr>
<td>测试井 (50µM)</td>
			<td>94</td>
			<td>22</td>
			<td>21</td>
			<td>2</td>
			<td>0</td>
			<td>0</td>
		</tr>
<tr>
<td>测试井 (100µM)</td>
			<td>86</td>
			<td>25</td>
			<td>21</td>
			<td>6</td>
			<td>1</td>
			<td>0</td>
		</tr>
</tbody></table>表 1: binning 不同屏幕的代表性结果 在这里, 我们提出了 binning 的平均百分比活分数从两个屏幕 (重新测试和候选)。在重新测试屏幕中, 峰值与负控制类似, 在表的右侧 (长寿命) 一侧, 再测试优于负控制。这表明重新测试集中存在多个支持寿命的化合物。在候选屏幕中, 我们看到峰值位于所有死类别中, 而表的右侧也有一个明显的信号。这表明在候选集合中存在的亲长寿化合物, 但也表明在候选化合物集合中存在显著的毒性。

Watch this Scientific Journal Video about A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans at JoVE.com

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

在这里, 我们描述了一个简单的协议, 快速生产数以百计的线虫生长培养基琼脂, 96 良好的文化板块, 其数量一致的Caenorhhabditis 线虫每井。这些文化对于整个有机体的表型筛选是有用的。我们专注于利用这些文化来筛选化学物质以促进长寿效果。

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

a-simple-method-for-high-throughput-chemical-screening-caenorhabditis

Research

JoVE Journal

Biochemistry

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

8.6K 次观看.  The Buck Institute for Research on Aging. 在这里, 我们描述了一个简单的协议, 快速生产数以百计的线虫生长培养基琼脂, 96 良好的文化板块, 其数量一致的Caenorhhabditis 线虫每井。这些文化对于整个有机体的表型筛选是有用的。我们专注于利用这些文化来筛选化学物质以促进长寿效果。

视频: A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

基于核磁共振的片段筛选，样品最少，自动化程度最高

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

一种简单的高通量化学筛选方法在秀丽线虫中的应用

摘要

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此视频中的章节

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

细菌内膜显示筛查抗体片段的图书馆

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

基于核磁共振的片段筛选，样品最少，自动化程度最高

活性秀丽隐杆线虫核提取物的分离及重建用于体外转录

一种简单的高通量化学筛选方法在秀丽线虫中的应用

摘要

探索更多视频

此视频中的章节

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

细菌内膜显示筛查抗体片段的图书馆

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

基于核磁共振的片段筛选，样品最少，自动化程度最高

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

活性秀丽隐杆线虫核提取物的分离及重建用于体外转录