我们要感谢托马斯Lecuit（SQH-的utrophin :: GFP线），布卢明顿库存中心，以及发育研究杂交瘤细胞银行试剂。我们还要感谢废话实验室全体成员的稿件有益的讨论和批评。从资助的美国国家科学基金会MCB-1158527，和启动从解剖学和细胞生物学系基金，爱荷华大学支持这项工作。健康博士前培训格兰特在药理科学T32GM067795国家机构支持AJS。数据存储支持通过信息通信技术，它是通过由国家研究资源中心和国家中心推进转化科学，国立卫生研究院，通过格兰特UL1RR024979支持CTSA提供资助。

1。准备果蝇在此之前隔振<ol><li>通过合并将50g活性干酵母用90毫升蒸馏水使湿酵母膏。混合用锅铲结合。让混合物静置约30分钟评估一致性之前。一致性应该是刚刚够厚坚持苍蝇小瓶的一面，但还没办下来的一面。以达到预期的一致性，可能需要添加最多加入10ml额外的水或少量干酵母。存放在有盖的容器中，在4°C。所存储的酵母可用于〜1-2周。 </li><li>收集&lt;24小时旧成蝇所需的基因型（男性和女性），并把它们与飞食品加湿酵母膏涂抹在小瓶的侧面小瓶。在该蝇维持温度将取决于实验的不同而不同。对于常规的实验，苍蝇都保持在室温。而实验，其中一个基因是使用UAS/GAL4系统15被过度表达可能需要维持蝇，在25℃或更高，适当的。 </li><li>每天提供苍蝇与酵母膏的涂抹新鲜的2天。注意：由于卵泡发育是由雌蝇的营养状况紧密调节，它是必不可少的，以提供即时的营养丰富的酵母膏为至少36小时之前剥离。 </li></ol> 2。隔离中旬至下旬上演果蝇卵泡<ol><li>允许介质来至室温（〜30分钟）。如果上演卵泡将要被用于在体外测定法的发展，新鲜制备IVEM媒体（Grace的昆虫媒介，10％热灭活的胎牛血清和1x青霉素/链霉素）。如果上演卵泡将要用于mRNA或蛋白分离使用任Grace的昆虫或IVEM媒体。重要提示：介质温度是至关重要的。如果媒体是冷的它会改变无论是肌动蛋白和微管细胞骨架和块的发展。 </li><li>通过注入CO 2气体麻醉苍蝇在yeasted小瓶中，并放置3-5雌性果蝇到飞垫CO 2条件下。重要提示：不要让更多的女性比可以在10分钟时间内被解剖上飞垫作为长期暴露在二氧化碳可引起不育和死亡。 </li><li>填补9点板与媒体之一好，把它放在一个黑暗的表面上（一块黑色有机玻璃效果很好）。专注于井底的解剖范围。 </li><li>使用＃5杜蒙钳拿起用一只手一个单身女性（这最好是通过使用惯用手）。它往往是最容易拿起女性的翅膀，腿，胸部或腹部来过渡。淹没女性在一个充满良好的媒体。调节显微镜的焦距是必要的。 </li><li>使用第二钳子在非优势手持，抓住女性在腹部的前端使后端被定位朝向优势手（ 图2A）。一定要保持淹没在媒体苍蝇。 </li><li>与惯用手，用钳子抓住角质层在第二最后色素段和撕离腹部（ 图2B-C）的其余部分。 </li><li>使用优势手的镊子，轻轻挤压腹部的前端，直到出现卵巢（图2D-E）。如果有必要，将镊子在两个卵巢之间以及拉出来的胴体。 </li><li>无论是移动卵巢一口新井用新鲜培养基（图2F）或取出尸体到的Kimwipe或纸巾。这是如果毛囊被隔离在体外发育尤为重要。 </li><li>重复，直到3-5对卵巢已被隔离。注：每个卵巢由〜16卵巢管或顺序成熟卵泡链。每个卵巢管包含在一个肌鞘。 </li><li>用针虎钳，并与他们提供的针，轻轻拉开卵巢通过运行卵巢管（ 图2G）的针。这有时会释放出单个毛囊为好。 </li><li>个别卵巢管和卵泡发育的很容易分辨形态的阶段现在应该是可见的（ 图2G，取笑除了卵巢）。参阅图1和图2H为可用于区分不同的阶段的形态差异。以分离的毛囊是完好卵巢管内，但仍包含在肌鞘，用针穿过卵巢管切割的前卵泡的前部，并在卵泡的后即刻感兴趣的卵泡以下。这将打破肌鞘和感兴趣的卵泡了，现在可以切离相邻的毛囊不会造成损坏。 </li&gt; <li>作为单独的阶段是分开的，将所感兴趣的卵泡，用玻璃移液管，以一个新的井用新鲜培养基。分离毛囊的体外发育时，这一点尤为重要。注意：在进入媒体，以防止气泡从再分配感兴趣的卵泡整个井前挤移液器灯泡是很重要的。此外，如果毛囊附着在玻璃吸管，该吸管可以通过吸取的BSA溶液上下数次并用解剖介质漂洗预涂用3％牛血清白蛋白（BSA）。 </li><li>一旦有足够的卵泡已被隔离，进行后续实验。 </li></ol> 3， 在体外发育S10B卵泡<ol><li>使用阶段隔离协议（步骤2）在IVEM介质隔离S10B卵泡。确保毛囊迅速远离碎片。由于这些毛囊会不断成熟，它的IMPOrtant该S10Bs收集30-60分钟，然后放置到所选择的成熟媒体。注：S10B需要被小心地从S10A卵泡（ 图1C相比，1B）区分开来，因为S10A卵泡不会在IVEM培养基成熟。在这两个S10A和S10B卵泡，一半长度的由护士细胞，而另一半是在卵母细胞，但在S10B毛囊的长度是等于该S14的卵泡。 </li><li>使用玻璃移液管中，将约30 S10B卵泡到24孔组织培养板的孔中。注意：请务必确认所有正在传输的卵泡是S10B，并没有成熟到S11。 </li><li>准备1微升成熟的媒体，每孔， 即 IVEM媒体加药理学试剂。 </li><li>使用拉玻璃移液管*，从油井（步骤3.2）中删除尽可能多的媒体的可能，并迅速补充选择成熟的媒体。注：必须使用一个拉玻璃点子ETTE作为阶段性的卵泡可以很容易地采取了两种玻璃吸管或移液器吸头用的Pipetman。 *为了使拉移液管：1）加热的长，玻璃移液管在本生灯的火焰中的薄的部分只是直到玻璃开始软化; 2）紧接在使移液管出来的火焰和拉水平地画出吸管进更细管; 3）打破生成一个细点。注意：使用保护眼睛的玻璃碎片可能飞了出去。 </li><li>尽可能多井重复步骤3.1-3.4的实验需要。 </li><li>让卵泡制定&gt; 10小时，根据得分解剖范围的发育进程。注：实验通常打进第二天，以便有足够的时间使卵泡发育。 S10B是〜5小时，S11是〜30分钟，S12是〜2小时，和S13是〜1小时，持续时间在25°C 1和发展在室温下需时稍长。类似的实验，以对于S10B卵泡描述可使用S11-S13毛囊进行。 </li></ol> 4。舞台隔离实时成像<ol><li>隔离后期阶段的卵泡（S10B-14）表示所选择的荧光标记物使用阶段隔离协议（步骤2）在IVEM媒体，快速移动的卵泡远离碎屑。 </li><li>移动感兴趣的毛囊进入新媒体，然后通过玻璃吸管转移到盖玻片底培养皿。小心，这样它泡在盖玻片底部区域，但不会波及添加介质。 </li><li>对于较长的时间推移的电影，有时需要通过增加一个卷起的Kimwipe，用水润湿，向培养皿的内侧边缘，以保持培养皿内的湿度。将盖在上面。 </li><li>图像上的倒置显微镜。详细解析将需要共聚焦显微镜。这是必要的，以平衡成像，照明的强度，并且成像的长度的频率，这将需要被独立地计算出每个升abeling工具和发育过程。 </li></ol> 5。舞台隔离的mRNA的制备<ol><li>使用阶段隔离协议（步骤2）无论是Grace的或IVEM介质隔离各个阶段的卵泡。 </li><li>用玻璃吸管，将兴趣变成新井与媒体的各个阶段，它可以收集多个阶段一次。 </li><li>继续收集时间在1小时。移动卵泡，用玻璃移液管，1.5 ml离心管中。 </li><li>简要地旋管在微型离心沉淀所有的卵泡。使用拉玻璃移液管（见步骤3.4），小心地取出所有介质。注意：如果使用的是全尺寸的离心，离心卵泡在低速。 </li><li>加入100μlTrizol法和手工研磨的〜20秒使用塑料杵。在微量降速全速移动的Trizol到一个新的1.5 ml离心管中，小心不要打扰任何沉淀ðebris。储存在-80℃。 </li><li>直到有足够的卵泡已为实验获得重复步骤5.1-5.5。通常，〜75 S10B，〜75 S12，和100〜S14的毛囊产生〜10微克的RNA。 </li><li>在冰上解冻样品。结合样品（如适用），成一个离心管中，并把Trizol试剂量高达800微升。与RNA分离进行所指示的制造商。重要提示：请记住，DNA酶处理分离的RNA与RNA酶的DNA酶自由。 </li></ol> 6。隔离阶段免疫印迹<ol><li>预热的加热块到100℃。 </li><li>使用阶段隔离协议（步骤2）无论是Grace的或IVEM介质隔离各个阶段的卵泡。注：这是必要的，以经验判断需要多少的特定阶段的卵泡，观察每一个特定的蛋白质。对于高表达的蛋白质1-3 S10B卵泡/孔也足以看出在免疫印迹的强烈信号。但是，它提出的是一个重要的代表性的样本，同时从多个女性卵泡。因此，毛囊15-20特定阶段的通常收集。 </li><li>移动卵泡，用玻璃移液管，到1.5ml微量离心管中。在微型离心旋转简要沉淀的卵泡（参见步骤5.4）。小心使用拉玻璃移液管取出所有介质的（见步骤3.4），并加入50微升的1×PBS和50微升的2倍Laemmli缓冲液。 </li><li>手工研磨的〜20秒用塑料杵。注：塑料杵可以重复用于磨削西方样品洗涤和热高压消毒。 </li><li>在热块煮沸样品10分钟。 </li><li>简要地对冰和旋全速15秒，微寒意。无论是立即加载到-20°C的SDS-PAGE凝胶或商店注意：如果样本存储，记得再沸样品装载到凝胶前。 </li><li>下列标准协议进行免疫印迹分析。 </li></ol>

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没有什么可以透露。

果蝇卵泡是由只有少数细胞类型，使得它非常适合于形态学和分子分析。此外，由于卵巢的结构，它是比较容易获得大量的卵泡发育的特定阶段的一个共同的解剖范围和最少的培训。由于每个阶段代表一个短的时间窗口，舞台隔离可以提供显著分子见解在那个阶段发生的发育过程。例如，我们使用中期到晚期滤泡隔离表征S10B，S12，和S14 18过程中出现的基因表达的变化。此分析显示...

每个果蝇卵巢由〜16卵巢管，或依次成熟卵室或滤泡链。每个毛囊是由一个单一的卵母细胞，15种系衍生的护士或支持细胞，称为卵泡细胞（ 图1A）〜650体细胞。 果蝇卵子发生划分为14个形态学定义开发1阶段。卵泡发育的各个阶段是在一个苍蝇看到了很多次，使它相对容易隔离的特定阶段的卵泡有相当数量。 卵子发生（阶段10B-14）的中期到晚期是特别适合于隔离阶段（ 图1）。在第一阶段10B（S10B），卵泡完全伸长（ 即其长度等于一个阶段14（S14）卵泡，参见图1和图2H）和毛囊的长度的一半是由护士细胞而另一半是卵母细胞（ 图1C）。在这个阶段，护士细胞发生戏剧性的肌动蛋白重塑，加强皮层肌动蛋白和产生微丝2的平行束。在同一时间，卵泡细胞群，称为心细胞，在迁移护士细胞和卵母细胞之间，和卵泡细胞2背团成为指定接受迁移以形成背侧附属物，管状呼吸装置的胚胎3 。护士细胞，然后合同（S11），其细胞质内容挤进卵母细胞在一个叫护士细胞倾销过程中，它提供了与卵母细胞所必需的因素，它完成胚胎发育（ 图1D）。护士细胞，然后经历细胞死亡（S12-S13）4，和卵泡细胞分泌和图案蛋壳5（ 图1E-G）。因此，卵子发生的到底是富有重要的发展ð形态发生过程。 分离的中期到后期阶段的卵泡（S10B-S14），可以用于各种用途，包括分子分析。例如，mRNA的上演卵泡可以分离用于RT-PCR，微阵列，或RNA定序分析。这使得一看基因表达在短期内发展的窗口，与目前只有少数细胞类型，并确定如何基因表达受任何药物或遗传扰动改变。级分离，也可用于通过Western印迹来看待蛋白质。这种分析是很重要的，因为它允许一个量化蛋白表达水平与野生型相对于在特定阶段的突变体。虽然人们可以使用免疫荧光分析，以达到类似的结果，荧光定量是由于严格的要求，所有的像素被检测6的线性范围内较不稳健。此外，免疫印迹分析可以提供其它信息，例如一个s如果蛋白质被翻译后修饰，或者被从一个特定的剪接同种型表达。分离的阶段，也可以用于进一步的纯化蛋白，包括亚细胞分级或免疫共沉淀。 阶段特异性毛囊分离，也可用于在体外测定法的发展7和实时成像8。隔离S10B-S13卵泡会继续发展到S14在简单的培养基（见下文）。要注意，S10A毛囊不会通过护士细胞进度使用本手稿讨论的培养条件下倾倒是很重要的。我们已经使用S10B 体外发育检测，以确定前列腺素的作用，既药理学和基因，在通过护士细胞倾销与发展为已读奏7,9调节肌动蛋白重塑。同样，开发的后期阶段也可以被分离，以确定药物治疗或基因的人的影响ipulations在特定的过程，如心细胞迁移，背附属物迁移/ 10的形成，和护士细胞死亡。此类测定法可用于执行显性交互屏幕或测定，例如，在杂合子的PXT或中fascin单独突变对S10B 体外发育没有任何效果，从双重杂合子展览护士细胞倾倒缺陷和在发展9块卵泡。 此外，由于S10B-13能在文化发展，都在这段时间发生的过程中，可以通过实时成像观测。这种成像可以简单地使用透射光（如果有一个只关心总的变化形态）或共聚焦显微镜利用转基因果蝇表达的荧光探针或滤泡沾上实时成像染料进行。实时成像被用来大大促进我们的发展过程的理解。事实上，住IMAGIN克的晚期阶段的卵泡不断扩大背侧附属物迁移的知识，小管10的一个例子。我们预计，额外的后期处理，包括护士细胞倾销过程中肌动蛋白动力学，实时成像将提供新的见解这些发育事件。需要注意的是，虽然S10A和卵泡发育的早期阶段就不会继续发展成S14的文化，活在成像过程中发展的阶段发生的事件有可能使用的替代培养条件11-14（见讨论更重要的是信息）。 在这里，我们提供详细的协议进行隔离晚期毛囊无论是在体外发育和生活成像，或分子分析（mRNA和蛋白分离）。

<table border="0" cellpadding="0" cellspacing="0" width="1098">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:253px;">Active Dry Yeast</td>
			<td style="width:195px;">Genesee Scientific</td>
			<td style="width:227px;">62-103</td>
			<td style="width:424px;">Any source of Active Dry Yeast is fine</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Grace&#8217;s Insect Media</td>
			<td>Lonza</td>
			<td>04-457F</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat Inactivated Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11050H</td>
			<td>Any Heat Inactivated FBS should work</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10x Pen/Strep</td>
			<td>Gibco/Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pin Vises and Needles</td>
			<td>Ted Pella, Inc.</td>
			<td>13561-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Spot Plate, Nine Well</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">#5 Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">24 multi-well plates</td>
			<td>Becton Dickinson</td>
			<td>35 3226</td>
			<td>Any 24-well tissue culture dish should work</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coverslip Bottom Dishes (35mm)</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Coverslip thickness will depend on the microscope/objective being used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glass pipettes</td>
			<td>Corning</td>
			<td colspan="2">7095B-5x (for transferring follicles)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glass pipettes - long</td>
			<td>Corning</td>
			<td colspan="2">7095B-9 (for producing pulled pipettes)</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Sample pestle (1.5 &#956;l; RNase/DNase free)</td>
			<td>Research Products International</td>
			<td>199228</td>
			<td>Any plastic pestle that fits 1.5 &#956;l microfuge tubes can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td></td>
		</tr>
	</tbody>
</table>

the utility of stage-specific mid-to-late drosophila follicle isolation

果蝇卵子或卵泡发育已被广泛用于促进复杂的发育和细胞生物学过程的理解。此方法主要介绍如何隔离中期到晚期滤泡（舞台10B-14），并利用它们来提供新的见解时的发育时间紧窗户发生的分子和形态学的事件。分离的毛囊可用于各种实验技术，包括在体外发育分析，实时成像，基因表达分析和蛋白质印迹分析。在卵泡期10B（S10B）或更高版本将完成发展文化，这使得一个基因或药物扰动结合体外发育定义这样的操作对开发过程中的特定时期发生的过程的影响。此外，由于这些卵泡发育，在文化，它们非常适合于实时成像研究，这往往表明调解形态事件的新机制。分离的毛囊也可用于分子分析。例如，在基因表达的变化所导致的遗传扰动可以被​​定义为特定的发育窗户。此外，在卵泡发育的特定阶段的蛋白质水平，稳定性和/或翻译后修饰状态可以通过蛋​​白印迹分析检查的分析。因此， 果蝇卵泡阶段特异性的隔离提供了一个丰富 ​​的信息源纳入发展和形态的广泛保守的进程。

当分离果蝇卵泡发育的特定阶段，必须能够准确地分辨出不同的形态阶段。这是S10A和S10B颇具挑战性，因为护士细胞和卵母细胞的每个占用毛囊长度的一半在这几个阶段（ 图1B相比， 图1C）。然而，S10A毛囊长度短于S10B卵泡，作为S10B毛囊充分拉长，从而在长至S14毛囊（ 图1C相比1G）相等。此外，毛囊或体细胞在所谓向心力毛囊细胞的卵母细?...

Watch this Scientific Journal Video about The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation at JoVE.com

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

中旬至下旬果蝇卵泡舞台专用的隔离是用于各种用途。这类卵泡发育中培养，其允许基因和/或药理学操作成与体外测定法的开发和实时成像。此外，毛囊，可用于分子生物学研究，如分离mRNA和蛋白。

的效用舞台专用中旬至下旬<em&gt;果蝇</em&gt;卵泡隔离

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

the-utility-stage-specific-mid-to-late-drosophila-follicle

Research

JoVE Journal

Biology

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

11.2K 次观看.  University of Iowa Carver College of Medicine. 中旬至下旬果蝇卵泡舞台专用的隔离是用于各种用途。这类卵泡发育中培养，其允许基因和/或药理学操作成与体外测定法的开发和实时成像。此外，毛囊，可用于分子生物学研究，如分离mRNA和蛋白。 

视频: The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

有针对性的表达绿色荧光蛋白在头发上使用体外病毒转导的卵泡

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

科研

生物学

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

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 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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

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  ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

新生儿肝外胆管的隔离

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...