我们感谢博士。裕农一月和韦斯Grueber提供在这项研究中所使用的飞股票。作者承认托马斯F和支持这项研究的凯特米勒Jeffress纪念信托（DNC）和乔治梅森大学教务办公室（EPRI）。

果蝇外周神经元的磁性珠排序总评（共完成“议定书”的时间：2.5-3小时） 在任何时候都保持一个干净，核糖核酸酶自由的环境标准的实验室程序必须遵守，以防止RNA降解。 当解剖果蝇幼虫角质层，并放置在细胞内分解缓冲区，外周神经元是脱离角质层的细胞之一。我们必须利用这个属性，并设计了这个协议，以去除大部分从非特异性的细胞，如肌肉和脂肪隔离DA能神经元前角质层。 通过实践，可以成功地完成了整个协议，大约2.5小时内。在实验开始前，必须完成编制抗体包被的磁珠。 1。准备装订细胞磁珠： 实验开始前，必须完成这一步。可以准备准备珠和储存在4℃直至需要。 <ol><li> 100μL磁珠M - 280链霉亲和素包被的磁珠在PBS的3倍，由500μL新鲜PBS resuspending制粒在一个强大的磁场，它每次清洗。 </li><li>最后直接在未经稀释的生物素标记的鼠抗鼠CD8a抗体（抗体浓度为100微克/毫升）100μL重悬磁珠。 </li><li>孵育1小时偶有轻度振荡，以防止沉淀冰的混合物。 [1μL磁珠M - 280可绑定0.05-0.10微克生物素抗体]。 </li><li>珠抗体混合物洗净三次步骤1.1中所述，以除去多余的抗体。现在涂有抗体的磁珠，并随时可以使用。商店珠抗体混合物在100μL的1X PBS在4 ° C，直到使用。 </li></ol> 2。选择和洗涤幼虫：（10-15分钟） <ol><li>挑选30-50年龄匹配的三龄幼虫，并放置在1.6毫升离心管1-1.2毫升1X磷酸盐缓冲液（PBS）。 （3-5分钟） </li><li>关闭管各1秒和涡最大设置三次。 </li><li>使用火抛光的巴斯德吸管完全弃上清。重复洗（2.1），涡街（2.2）步骤3-4次，直到上清液明显清晰的任何食物颗粒和碎片。 </li><li>简单重复1毫升70％的乙醇洗，弃去上清液。 </li><li>用1毫升的ddH2O洗两次幼虫和上清液。 </li><li>简单重复洗一次1毫升的RNase的上清液。 </li><li>洗净的幼虫在1毫升的ddH2O三次，以确保彻底清除核糖核酸酶的。 </li></ol> 3。解剖：（10-12分钟） <ol><li>广场10-12幼虫上的一个Sylgard涂层35毫米的Petri板中心。位置稍微远离对方。 [ 关键的一步：放弃任何，似乎没有要在适当的发育阶段的幼虫] </li><li>使用所有的幼虫幼虫一双细清扫剪刀剖开前提示。 </li><li>使用一对沉闷杜蒙反转幼虫内而外的第5号钳。幼虫角质层内插入之一forcep所有后结束。捏钳提示一起抢的角质层（图1a）（尝试按Sylgard表面的角质层，使其更容易）后结束。使用产钳的第二对，推动内而外的幼虫角质层。 [ 关键的一步：尝试练习这种方法，细胞的分离实验，然后再尝试几次。设法得到完全倒置的幼虫，以确保所有的软组织暴露容易分解的解决方案。] </li><li>解剖3-4幼虫后，他们立即转移到新鲜的，冰冷的PBS（管放在冰）在1.6 ml离心管。 </li><li>重复步骤3.1至3.4，直到所有需要的幼虫收集（30-40幼虫在本议定书）。 [ 关键的一步：预计10-20％的亏损，在解剖和离解，并计划据此] </li></ol> 4。去除松散附着的非特异性细胞：（2-3分钟） [这一步艾滋病松散贴壁的非特异性的组织，如脂肪机构和中枢神经系统的结算。] <ol><li>取1.6 ml离心管中倒幼虫表皮及更换约700-800新鲜冰冷的PBS液的上清液。 </li><li>脉冲涡流离心管5倍全速每脉冲（3秒）。 </li><li>弃上清，约700-800μL新鲜，冰冷的PBS代替。 </li><li>重复步骤4.2和4.3的三倍。 </li><li> RESuspend幼虫表皮在400μL新鲜的，冰冷的PBS </li></ol> 5。离解成单细胞悬液，组织：（18-20分钟） [ 关键的一步：“过度分离可能导致导致不良的细胞产量和细胞存活率低的细胞表面标记的损失。幼虫组织可以分离，无论是机械分离（超声，douncing），酶解（胰蛋白酶，胶原酶等）或两者的结合。由于这些幼虫组织难以解离，我们发现，机械和酶解相结合产生了最好的结果。] <ol><li>添加1.5μL1X Liberase Blendzyme 3（28瓦nsch单位/瓶）400的PBS液中悬浮的幼虫角质层。 </li><li>涡的解决方案为1秒的最大设定值（这应该到解决方案中删除从角质层松散贴壁细胞）每2-3倍。 </li><li>孵育5分钟，在室温（22-25℃）的解决方案。 [ 关键的一步：孵化时间极大地影响了最终的细胞分选效率。建议的孵化时间应足以松动组织。不要超过15分钟。] </li><li>脉冲涡流管，最大设置为2秒每脉冲全速20-30倍。这应该释放到溶液中的肌肉和其他组织。检查一个小样本，从一个荧光立体显微镜下，在每一步的解决方案。 （有了经验，应该能够确定的解离程度，直接启用的荧光立体显微镜下观察离心管） </li><li>幼虫表皮洗净，用新鲜的，冰冷的PBS的2-3倍，并终于重悬于500μL新鲜的，冰冷的PBS含1％BSA。 </li><li>为了避免幼虫坚持2毫升Kontes组织磨床和大间隙杵，预涂层组织磨床和杵在1％BSA的PBS液和经过短暂的冲洗丢弃BSA溶液的玻璃表面的角质层。随后，使用火抛光的巴斯德吸管，转让步骤5.5角质层BSA的涂层组织磨床。 [ 关键的一步：预冷组织磨床/上放置了几分钟的冰，以防止细胞损伤/裂解杵] 。 </li><li> Dounce与缓慢而稳定的笔触的组织，避免起泡（约20-30杆）。 [ 关键的一步：Dounce缓慢和稳定，否则细胞裂解] </li><li>为了评估细胞分解水平，擦拭用干净的Kimwipe组织的组织磨床的外墙，和一种荧光立体显微镜下检查。应该有脱离的神经元的角质层，和解决方案中可以看出。如果发现这是很难，或者吸管出一个解决方案的小样本，和一种荧光立体显微镜下观察。细胞分解的一个很好的迹象是从幼虫角质层的神经元的情况下。但是，如果仍然观察连接到角质层的神经元，或观察不完全分离的细胞，进一步dounce，待细胞实现单细胞悬液。 </li><li>磨碎的解决方案，用火抛光的巴斯德吸管5倍缩小到标准的尖端直径约50％，其次是用火抛光的巴斯德吸管10倍缩小到标准的尖端直径约25％。 [ 关键的一步：有力trituration可能会损坏细胞。监控步骤之间的细胞，并调整相应的程序]。 </li><li>通过一个30微米的细胞过滤器的过滤解决方案，并在1.6 ml离心管收集细胞滤液。最终的解决方案应该由一个单细胞悬液，现在准备磁性细胞分选。 </li></ol> 6。磁珠细胞分选：（45 - 75分钟，依赖于抗体孵育时间） <ol><li>加入15μl抗体包被的磁珠的细胞悬液500μL（步长5.10）。余下的抗体偶联的磁珠可存储，直到随后的细胞隔离需要。 </li><li>抗体包被的磁珠细胞孵育冰偶尔手工搅拌30-60分钟。 关键的一步：在较高的温度或较长时间的潜伏期，可能会导致非特异性抗体结合。 </li><li> 2分钟的离心管中，放置在一个强大的磁场。正选择以及未绑定的珠所有细胞区分开来管方。 </li><li>慢慢地移液管取上清液，确保不打扰细胞沉淀。 </li><li>新鲜的，冰冷的PBS清洗细胞3-4次，以消除任何剩余的非特异性细胞。 </li><li>在30μL新鲜的，冰冷的PBS重悬细胞。 </li><li>近似​​的细胞的纯度和产量，吸取5μL的细胞悬液ension一个血球的表面抛光和一种荧光立体显微镜下可见荧光细胞计数。同时检查的非荧光的细胞和杂质的任何迹象量。通常情况下，样本将被高度浓缩的荧光细胞。 </li></ol> 7。磁性微球的排序细胞RNA的提取：（60 - 75分钟） <ol><li>计数，颗粒在磁场的细胞后，弃去上清液，并添加PicoPure™RNA分离试剂盒（Molecular Devices公司）20μL提取缓冲液。根据细胞数量，可能需要更高的提取缓冲液中添加的量。 </li><li>旋涡管的最大速度，使细胞沉淀提取液混合。 </li><li> 30分钟，42℃孵育管。 </li><li>为了确保搬迁前柱纯化的RNA（见第7.5步）的磁珠，简要离心管是在2000（X）G为2分钟沉淀磁珠。管，然后放置在一个强大的磁场，保留沉淀，上清液转移到一个新的离心管。 </li><li>提取和柱纯化的RNA PicoPure RNA提取试剂盒制造商的指示。 DNA酶处理是可选的，可以列上的RNA纯化过程中，根据分析要求。最后，洗脱，直到准备使用绑定在一个小体积的洗脱缓冲液并储存于-80 ° C（11-30μL）的总RNA。如果需要一个1μL等份可用于评估一个2100生物分析仪（安捷伦科技公司）的总RNA质量。 </li></ol>代表性的成果： 磁珠排序是用来隔离果蝇 DA能神经元（图1）。从这些孤立的DA能神经元（图2a）纯化的RNA被发现的优秀品质急剧5.8S，18S和28S核糖体RNA峰的存在表明，在Agilent 2100生物分析仪（安捷伦科技公司）进行分析时，（图2b）。 30-40第三龄幼虫开始，我们平均300-500类IV DA 使用PPK - GAL4驱动神经元和1500-2000 DA能神经元（I类，II，III及IV）使用隔离的泛DA神经元的具体GAL4 21-7驱动程序。为了评估特定的浓缩，分离的细胞神经元，我们进行了定量逆转录聚合酶链反应（QRT - PCR），使用两个神经元的基因特异性标记（ elav和futsch）。这些分析显示显着的两个标记基因相比，通过使用我们的协议（图3）流动表明DA能神经元的一个非常具体的浓缩倍浓缩。最后，来自泛DA能神经元和第四类DA能神经元的分离出的RNA是用于执行安捷伦果蝇全基因组寡核苷酸芯片（4 × 44K）（图4）的转录表达谱。这些分析确定了另外一个以前未知的分子和公认的信号转导通路可能DA神经元的发展发挥重要的职能作用的广谱的DA神经元枝晶形态发生许多以前牵连的监管机构。目前正在进行研究，设计，评估的潜在作用，这些以前未知的分子（S）在调解DA神经元的发展，特别是枝晶的形态发生。 <img src="/files/ftp_upload/1599/1599figure1tmb.jpg" border="0" /> 图1： 果蝇 DA能神经元的磁性珠排序示意图（一 ）年龄匹配的第三龄幼虫轴承的DA神经元特异性GAL4，记者UAS - mCD8 - GFP转基因是由反相幼虫角质层内而外，揭露解剖PNS的解离缓冲区，并存储在冰冷的PBS（二 ）进行酶解加入Liberase Blendzyme 3的溶液中含有幼虫的角质层（三）幼虫组织进一步震荡，trituration分离和douncing除去非特异性标记的组织，如脂肪组织，内脏和中枢神经系统（D，E）细胞，然后使用30微米的细胞过滤器过滤。该解决方案包含了不同类型的细胞，包括上皮细胞，肌肉和神经元的单细胞悬液。，（F）抗鼠CD8a抗体涂层磁珠M - 280加入细胞悬液，冰浴30-60分钟。（ G）的磁珠结合的DA能神经元表达的标记GFP融合蛋白（H，I）的磁珠涂层细胞是由放置在一个强大的磁场解决方案分离的CD8 +鼠标。上清液丢弃，细胞洗3次，以除去残留的非特异性的细胞，从而导致（ J）Highly纯化DA能神经元的数量。请<a href="https://www.jove.com/files/ftp_upload/1599/1599figure1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1599/1599figure2tmb.jpg" border="0" /> 图2：（一）积极选择，绿色荧光蛋白荧光类IV DA神经元细胞分离和磁珠排序隔离的形象代表。导致神经元的人口被确定为高度富集类IV DA能神经元很少或没有污染的细胞杂质。隔离磁珠排序的大神经元（B），安捷伦2100生物分析仪（安捷伦科技公司）的总RNA电泳，表现出一个优秀的总RNA质量存在5.8S，18S，28S rRNAs。请<a href="https://www.jove.com/files/ftp_upload/1599/1599figure2.jpg">点击这里</a>看大图图2。 <img src="/files/ftp_upload/1599/1599figure3.jpg" border="0" /> 图3：QRT - PCR分析神经元的标记基因的表达在孤立的DA能神经元（GAL421 - 7，UAS - mCD8 - GFP），并通过一小部分的流量是一式三份。 QRT - PCR的两个特定神经元的标记基因（elav和futsch）的表达水平进行了评估。从这些分析中得到的值进行标准化的内对照（rp49），和相对流量的一小部分通过观察这些水平计算ΔΔCτ 方法 6 。 elav和futsch都极大地丰富DA神经元中分离出来的人口相比，流动通过分数。 <img src="/files/ftp_upload/1599/1599figure4tmb.jpg" border="0" /> 图4：代表类第四大神经元特异性Cy3标记，标记的微阵列图像文件。这里展示的是安捷伦的果蝇全基因组寡核苷酸芯片（4 × 44K）与Cy3标记的labeld类第四大经磁珠排序纯化的神经元分离的总RNA杂交。图4看到一个更大的版本， <a href="https://www.jove.com/files/ftp_upload/1599/1599figure4.jpg">请点击</a>这里。

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这里介绍的协议优化，坚持紧紧地使用磁珠细胞分选策略果蝇三龄幼虫角质层内表面的外周神经元的分离和纯化。虽然我们已经使用了此协议专门隔离果蝇DA能神经元，此协议的应用到其他类型的细胞，角质层中的幼虫或蛹的发展阶段，坚持以隔离（如上皮细胞，肌肉，其他的外周神经元）可适应由不同的几个参数，并使用不同的GAL4，记者UAS - mCD8 - GFP转基因标签的细胞类型或类型的利益。...

设备<ul><li> （10）钕块磁铁（K＆J磁公司，卡特彼勒。B444B），或者DynaMag™-2（Invitrogen公司，卡特彼勒。＃123 - 21D），也可使用。 </li><li> Kontes玻璃组织研磨机，2毫升的工作能力，与大间隙杵（货号＃885300-0002） </li><li>细胞过滤器，如磁珠预分离过滤器（美天旎生物技术公司，卡特彼勒＃130-041-407） </li><li> 35毫米培养皿菜涂有Sylgard（道康宁公司，卡特彼勒＃3097358-1004） </li><li>解剖工具：两对杜蒙号5钳（精细的科学工具，猫＃11251-20。） </li><li> Vannas春天微解剖剪刀（精细的科学工具，猫＃15000-08） </li><li>配有一次性尖头滴管（P - 1000 P - 100 P - 10） </li><li>标准血球计数细胞和估计纯度</li><li>与标准直径的火抛光提示巴氏吸管〜50％的标准直径，缩小和收窄至〜25％的标准直径为trituration </li><li>台面微型离心机（1-16,000（X）G） </li><li>涡旋</li><li>荧光立体显微镜（徕卡MZ16FA在本议定书） </li></ul>

isolation and purification of drosophila peripheral neurons by magnetic bead sorting

“<em&gt;果蝇</em&gt;周围神经系统（PNS）是一个强大的调查功能和分子水平的神经元发育和树突形态发生的复杂过程模型。为了帮助这些分析，我们已经开发出一种称为树突分枝（DA）神经元，已被广泛用于研究树突形态发生的PNS的神经元的一个子类的隔离策略<sup&gt; 1,2</sup&gt;。这些神经元是非常困难的隔离作为一个纯粹的人口，部分原因是其极低的发生，他们难以达到低于强硬的几丁质的幼虫角质层位置。我们新开发的方法克服了这些挑战，是一个快速和特定的细胞抗体包被的磁珠富集。磁珠分选的研究，我们已经使用了年龄相仿的三龄幼虫，表达了小鼠CD8标记的GFP融合蛋白（<em&gt;无人机mCD8 - GFP</em&gt;）<sup&gt; 3</sup&gt;控制下的IV级树突分枝（DA）神经元特异性<em&gt;扒手（PPK）- GAL4</em&gt;驱动程序<sup&gt; 4</sup&gt;或控制的泛- DA神经元特异性GAL4<sup&gt; 21-7</sup&gt;驱动程序<sup&gt; 5</sup&gt;。虽然此协议已被隔离PNS的不同的几个参数，连接到幼虫角质层的内壁细胞优化，可以使用相同的协议，以分离出许多不同的细胞类型，幼虫或蛹角质层发展（如上皮细胞，肌肉，oenocytes等），或从GAL4特定的驱动程序的表达模式后的幼虫机关的其他类型的细胞。用这种方法分离出的RNA质量高，可以很容易地用于下游基因组分析，如微阵列基因表达分析研究。这种方法提供了一个强大的新工具来进行孤立的研究<em&gt;果蝇</em&gt;树突分枝（DA）神经元底层树突形态发生的分子机制，从而提供了新的见解。

Watch this Scientific Journal Video about Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting at JoVE.com

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

在这个视频文章中，我们目前的分离纯化的方法<em&gt;果蝇</em&gt;使用快速磁珠辅助细胞排序策略外周神经元。从分离的细胞获得的RNA可以很容易地用于下游应用，包括微阵列分析。

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

isolation-purification-drosophila-peripheral-neurons-magnetic-bead

Research

JoVE Journal

Biology

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

13.3K 次观看.  George Mason University. 在这个视频文章中，我们目前的分离纯化的方法果蝇使用快速磁珠辅助细胞排序策略外周神经元。从分离的细胞获得的RNA可以很容易地用于下游应用，包括微阵列分析。

视频: Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

从外周血中分离和鉴定的微泡

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

分离纯化果蝇外周神经元