Name: live cell cycle analysis of drosophila tissues using the attune acoustic focusing cytometer and vybrant dyecycle violet dna stain
Uploaded: 2013-05-19T14:00:00
Description: Watch this Scientific Journal Video about Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain at JoVE.com

我们感谢阿依德拉克鲁兹的开发和教学的原始协议，这个版本是基于10。工作在Buttitta实验室是由美国国立卫生研究院授予GM086517支持。

1。飞畜牧<ol><li>交叉飞行所需的基因型在狭窄的塑料小瓶10毫升酵母葡萄糖培养基或其他富含蛋白质的媒体你的选择。详细4,5中描述的果蝇基因的广泛的工具可用于组织特异性表达和体内荧光蛋白表达的谱系追踪。父母每天获得24小时后代集合一系列新鲜小瓶，或更精确的分期，收集胚胎琼脂平板上，新孵出的幼虫转移到小瓶描述了10位。为了确保跨实验和统一的条...

<ol>
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这里描述的协议可以分析细胞周期，细胞相对大小和相对活果蝇在不同的发育阶段组织细胞数量。当这种分析是细胞类型特异性荧光蛋白表达谱系追踪再加上详细的信息，可以得到谨慎细胞周期或增长的扰动有关的细胞反应。作为证据的原则，我们在蛹飞脑扰乱了静止在GFP标记的细胞克隆表达G1-S细胞周期调控，导致异常的DNA复制处于发展阶段时，脑细胞通常超过90％的G1被捕（ 图3D）...

可用于测量细胞活力，电池的相对大小，DNA含量在活的细胞群的荧光蛋白表达的流式细胞仪。由于核DNA复制在S阶段，人口细胞DNA含量的信息可以用来推断相对百分比，在不同的细胞周期1-3。这种方法已经成为模型系统从酵母到哺乳动物细胞周期分析的基石。 果蝇基因在体内的细胞周期调控的分析已经成为一个很好的模型系统。苍蝇提供广泛的遗传工具允许优雅的组织特异性和时间调节细胞周期调控操作一起在体内的荧光蛋白质为基础的谱系跟踪4-6。流式细胞仪已被用于研究果蝇的一些类型的细胞中DNA的含量，包括endorepl指令，这是通过将细胞和培养的细胞有丝分裂专用7,8。德拉克鲁兹和埃德加是由体内细胞周期研究的一个重要进展，现场二倍体果蝇成虫盘9,10流式细胞仪分析，协议已经被许多实验室使用和修改的协议的发展。该技术中，加上通过诱导荧光蛋白表达和组织特异性标记基因在体内的谱系追踪，允许一个取得整体细胞倍增时间，细胞大小的基因操作的影响的信息，并确定细胞周期的各个阶段的精确的定时， 在体内 9 ，11。然而，这种方法迄今依赖于使用的细胞可渗透的Hoechst 33342 DNA嵌入染料染色和定量活细胞中的DNA，这限制了用户流能够令人兴奋的Hoechst染料的UV激光器的流式细胞仪。这些通常是发现，只有在分拣机（ 即 BD FACS宏达GE，BD FACSAria）或昂贵的多色台式系统（ 即 BD LSR），通常需要支持机构流动的核心设施。 我们已经修改了赫斯特为基础的协议，使用新的活细胞DNA染料从Invitrogen公司，Vybrant DyeCycle紫。这种染料是带有紫罗兰的405 nm激光，多见于较小的台式分析仪，可在小自足的台式分析仪，Attune的声波聚焦流式细胞仪兼容。在这里，我们提出了一个详细的协议分析细胞周期，细胞类型，细胞大小，细胞数量和谱系分析在各种果蝇组织在不同的发展阶段使用DyeCycle紫上调，可以配上。该协议扩大了流式细胞仪适合进行这种分析与果蝇组织和提供这种类型的活细胞周期分析如何可以修改为其他组织类型和发展阶段的例子。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>12x75 mm Polystyrene Round-Bottom 5 ml Test Tube </td>
 <td>BD Falcon </td>
 <td>352058 </td>
 <td>5 ml tubes</td>
 </tr>
 <tr>
 <td>Attune Acoustic Focusing Cytometer</td>
 <td>Life Technologies/ Applied Biosystems</td>
 <td>4445315</td>
 <td>Blue / Violet configuration</td>
 </tr>
 <tr>
 <td>Attune Cytometer Software (version 1.2.5)</td>
 <td>Life Technologies/ Applied Biosystems</td>
 <td>Free </td>
 <td>PC only</td>
 </tr>
 <tr>
 <td>Attune Performance Tracking Beads (5 x 106 beads/ ml) </td>
 <td>Life Technologies/ Applied Biosystems</td>
 <td>4449754 </td>
 <td>For daily performance test</td>
 </tr>
 <tr>
 <td>Dumont #5 Inox forceps </td>
 <td>Fine Science Tools</td>
 <td>11251-20 </td>
 <td></td>
 </tr>
 <tr>
 <td>Embryo dishes 
 30 mm x 12mm </td>
 <td>Electron Microscopy Sciences</td>
 <td>70543-30 </td>
 <td>Glass dissection dishes </td>
 </tr>
 <tr>
 <td>Eppendorf Thermomixer</td>
 <td>Eppendorf</td>
 <td>022670051 </td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin-EDTA Solution (10x)</td>
 <td>Sigma</td>
 <td>T4174 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vannas-T&uuml;bingen Spring Scissors </td>
 <td>Fine Science Tools</td>
 <td>15003-08 </td>
 <td>Straight 5mm Cutting Edge </td>
 </tr>
 <tr>
 <td>Vybrant DyeCycle Violet Stain</td>
 <td>Life Technologies/ Invitrogen</td>
 <td>V35003 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Required reagents and instruments.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Live DNA Stain Solution (10 ml):
 1 ml 10X Ca2+ Mg2+ free PBS (pH7.2) 
 9 ml 10X Trypsin-EDTA (Sigma) 
 5 &mu;l Invitrogen Vybrant DyeCycle Violet (note that this is 0.25X the recommended concentration for mammalian cells. We find that higher concentrations are toxic to Drosophila cells.)
 10X Ca2+ Mg2+ free PBS (pH7.2): 1.37M NaCl, 27 mM KCl, 100mM Na2HPO4 (dibasic), 20mM KH2PO4 (monobasic) adjusted to pH 7.2
 </td>
</tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td><table border="1">
 <tbody>
 <tr>
 <td></td>
 <td>FSC</td>
 <td>SSC</td>
 <td>BL1</td>
 <td>VL1</td>
 </tr>
 <tr>
 <td>Threshold (x1000)</td>
 <td>100</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 </tr>
 <tr>
 <td>Voltage (mV)</td>
 <td>2950</td>
 <td>4250</td>
 <td>1800</td>
 <td>1150</td>
 </tr>
 </tbody>
 </table>
Table 2. Threshold and voltage setting for the analysis in Figure 2.</td>
 </tr>
 </tbody>
 </table>

live cell cycle analysis of drosophila tissues using the attune acoustic focusing cytometer and vybrant dyecycle violet dna stain

流式细胞术已被广泛用来获得的细胞群体中的DNA含量的相关信息，推断出在不同的细胞周期的各个阶段的相对百分比。此技术已成功地扩展模式生物果蝇体内的细胞周期调控的遗传研究有丝分裂组织。当加上细胞类型特异性荧光蛋白表达和遗传操作，可以得到对细胞数，细胞大小和细胞周期的相位在体内的详细信息。活细胞的方法，然而，这依赖于细胞渗透性的Hoechst 33342 DNA嵌入染料的使用，限制用户配备UV激光器的流式细胞仪。我们已经修改了协议，使用一个新的活细胞DNA染料，紫Vybrant DyeCycle，兼​​容比较常见的紫405nm的激光。这里介绍的协议允许高效加上细胞的细胞周期分析类型，电池的相对大小和细胞数的信息，在各种果蝇组织。此协议扩展有用的细胞周期分析技术在活果蝇组织中的一个小的台式分析仪，调到声波聚焦流式细胞仪，一个单一的实验室规模上可以运行和维护。

图2所示为一个幼虫翼样本的代表性的结果，组织的后半部，表达绿色荧光蛋白GFP模板。得到类似的结果具有相同的组织的类型和使用所提供的RFP模板（ 图3A）为RFP的表达模式。提供的模板和电压（表2）适合幼虫的眼睛（ 图3B），大脑和翅膀，以及蛹的眼睛，大脑（ 图3D）和翅膀的分析。盖茨5和6的人口密谋FSC提供相对单元尺寸也可以生成柱状图...

Watch this Scientific Journal Video about Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain at JoVE.com

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

活细胞周期分析的一种协议<em&gt;果蝇</em&gt;组织使用同调声对焦流式细胞仪进行说明。该协议同时通过提供信息相对细胞大小，细胞数量，DNA含量及细胞类型谱系跟踪或组织特异性表达的荧光蛋白<em&gt;在体内</em&gt;。

活细胞周期分析<em&gt;果蝇</em&gt;组织使用同调声聚焦流式细胞仪Vybrant的DyeCycle紫DNA染色

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Research

JoVE Journal

Biology

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

科研

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实时成像果蝇胚胎血细胞迁移

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凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

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Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

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The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...