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件，避免过度拥挤，F1代单瓶总数应不超过100。根据遗传的实验设计，在适当的孵化器，直到所需的发育阶段保持小瓶。 </li><li>收集实验动物:您将需要在第三幼虫老熟幼虫龄幼虫解剖准确的蛹分期或白色前蛹（WPP）（L3）约110-120小时的发展。戏剧细胞周期的变化，包括非增殖状态切换到发生在不同的时间点，在变态12,13。因此，正确分期蛹（1小时内的发展）重现的细胞周期的蜕变过程中的数据是必不可少的。 WPP对应为0小时后蛹的形成（0小时APF）在图1中所示，面板I采集WPP在一个35毫米的培养皿上的折叠的湿Kimwipe用2.5毫升水，以保持一定的湿度。年龄蛹，直到所需的阶段（小时APF）在适当的温度。需要注意的是发展收益1.2倍的速度在29°C和2.2倍，更慢，在18°C比标准的25°C 14。 </li><li>热休克动物谱系追踪克隆，如果需要9,11激活热休克flipase，（HS-FLP）诱导重组。对于幼虫，浸泡热休克完全浸没在水浴中（在37℃下），并确保幼虫的小瓶。热休克蛹，用封口膜密封的培养皿中，完全淹没到水浴。请务必除去封口膜的热休克后，动物的生存，以便有足够的氧气交换。热休克的持续时间依赖于活性flipase使用的转基因和所需的克隆数。我们经常使用诱导"FLIPOUT"谱系追踪克隆15，HS-FLP第一号染色体上的基因和2-4分钟的热休克蛹在35 mm培养皿幼虫7-8分钟，而HS-FLP转基因的第二染色体需要20分钟。热休克克隆获得类似的数字。 </li></ol>细胞分裂的速度和清扫时机决定在每个组织中克隆大小。在正常条件下，我们发现，克隆幼虫翼诱导热休克20分钟（hs-FLP的第二染色体上的转基因）和dissec泰德48小时后，包含约20-30大小不等，从每10个细胞克隆两个单元，每个克隆的分离克隆，平均大约每四个单元克隆。相比之下，7分钟0小时APF同一HS-FLP转基因蛹在培养皿中的热休克36小时后解剖，很好的分离克隆（约15-25）介乎一至四个单元的平均收益率2.2每个克隆细胞。这样的数据可以被用于确定所研究的组织的平均细胞倍增时间。 2。解剖<ol><li>轻轻的幼虫/蛹转移到一个透明的玻璃夹层菜含有1X PBS。 </li><li>使用解剖显微镜和充足的光，轻轻抓住幼虫腹部用锋利的INOX＃5镊子，微型剪刀剪前三分之一，并反转前三分之一推口区向后路向内按住幼虫角质层稳定（ 图1A）。小心删除opaqu时É脂肪体和任何剩余肠道所需翼，眼或脑组织，如提高知名度。解剖所需的组织;干净去除气管和角质层的翅膀从口钩，镊子，眼睛或脑眼（ 图1G）。 </li><li>解剖蛹;使用镊子轻轻抓住蛹的蛹壳的前尖那里是一个气泡，以避免损坏的蛹内（ 图1C）。中的相对的手钳或微型剪刀可以用来帮助定位浮动蛹进行适当的抓。干净微型剪刀划破后尖蛹。这种横截面的切应在一个很小的角度朝向动物的背侧部分，释放内部压力而不迫使histolyzed的脂肪转化为所需的组织中的胸部和头部。小心剪下沿背正中，避免机翼和前眼脑复杂。小心取出蛹，从蛹情况下抓住蛹表皮后缘，然后将其拉出的蛹的情况下（ 图1D）。角质层前眼脑复用钳子戳一个小洞。使用玻璃巴斯德吸管带有橡胶球，轻轻洗PBS解剖的解决方案，通过局部解剖蛹，删除histolyzed脂肪和任何剩余的肠道组织。这也有效地润滑与脂质和蛋白质的解剖组织的发生率，从而降低了从粘的玻璃移液管的内部购买的玻璃吸管。 </li><li>要取得蛹翅膀，角质层包围机翼和翼本身必须撬开从蛹（ 图1E）。这是可以做到保持蛹由磁头与镊子，同时使用另一种的镊子可以掌握翼角质层的最后的小费和拉出或机翼下的在铰链附近的操纵镊子从侧面撞出之前胴体（FIGURE 1E）。机翼可以在铰链处被剪开或撕开，从体内取出，并把在一堆远离屠体及其他材料。 </li><li>为了获得蛹的眼睛或脑部，洗蛹，直到眼脑复从蛹（ 图1F，1H）脱落和免费。使用镊子，轻轻一拉蛹的眼睛远离大脑和保存所需的组织。 </li><li>不要长于45分钟，每个样品解剖，结果变得不那么准确较长的细胞后，留在PBS开放的动物。 </li></ol> 3。组织分离和DNA染色<ol><li>使用润滑巴斯德吸管，仔细解剖组织转移到0.5毫升现场DNA染色溶液5毫升聚苯乙烯管（ 图1G）。转移到DNA染色溶液作为小PBS尽可能（小于300微升），会抑制胰蛋白酶的稀释组织分离。 </li><li>孵育管夏奇吴先生在23°C在500转（我们使用的Eppendorf恒温）。 </li><li>孵育70分钟，然后轻轻地，持续5秒，在中等速度（设定5）涡。过度的震荡会导致细胞破坏和碎片形式，而下的震荡会导致细胞团块。返回样品摇晃在23°C在500转一个额外的15至20分钟涡旋前一次的速度，持续5秒5。 </li><li>对于的头脑和蛹的眼睛比36hAPF旧的，修改协议是必需的。在23°C在500转游离组织中DNA染色溶液1.7毫升离心管中的45-60分钟。然后一个接一个手动分离大块组织转移到解剖盘，连同约50微升的DNA染色解决方案和快速上下吹打手工绘制的巴斯德吸管16（约100-150微米开口）。每个分离的试样转移到5ml的管0.5毫升的DNA染色溶液的其余部分。孵育45-30分钟（总孵化时间高达90分钟），在23℃以500rpm振荡，直到不再是可见的团块。 </li><li>完全分离的样品应该有极少数的大可见大块的组织，虽然注意到脱细胞透明蛹翼角质层不会分解。 </li></ol> 4。流式细胞仪<ol><li>打开Attune的流式细胞仪，并打开Attune的软件。流式细胞仪激光器应通过启动菜单中打开，应该有足够的分数进行绩效跟踪珠每一天前的数据采集和日常表现测试。液体水平应该是至少半满和废物的性能测试之前清空。启动，适当上调校准和操作的详情，可以发现在Attune的用户指南（ <a href="http://tools.invitrogen.com/content/sfs/manuals/cms_082577.pdf" target="_blank">http://tools.invitrogen.com/content/sfs/manuals/cms_082577.pdf</a> ） </li><li>设计一个新的TEmplate设置的参数在4.3中创建一个空白工作区中的4点图2直方图，或选择预先设计好的模板，适合进行试验，并表示要收集的样本数。我们已经提供了活果蝇细胞周期分析与Vybrant DyeCycle紫罗兰和GFP或RFP表达的附录一Attune的模板（。格式的文件） </li><li>提供的模板包括四个点的情节和两个直方图如图2所示的参数。检测渠道包括:FSC和SSC - 向散射和侧向散射，表明细胞的大小和膜的复杂性，VL1高度（H），宽度（W）和面积（A） - 表明紫激光激发紫DyeCycle染色的DNA，和BL1 - 蓝色激光通道1区，表明绿色荧光。本RFP的分析提供了模板GFP分析的模板是相同的，只是它使用蓝激光通道3（BL3-A），这是优化作业捷思RFP检测。 </li><li>在模板中的点图的大门，以限制到所需的种群分析和分析（ 图2），以限制碎片和细胞团块。 1号门不包括碎片和SSC与FSC（ 图2A）的基础上的团块。门2不包括未染色细胞，亚G1期的凋亡细胞和根据识别汗衫（VL1Area与VL1Width的）（ 图2B）的细胞团块。栅极3是一个派生的栅极，包含门1和2的细胞内，并识别绿色荧光蛋白（RFP）与阴性细胞DNA含量根据对VL1Height（ 图2C）。门4是一个派生的栅极，包含门1和2的细胞内，并确定了绿色荧光蛋白（RFP）与DNA含量的阳性细胞（ 图2C）。绿色荧光蛋白（RFP）与前向散射（FSC）所测得的细胞的大小的点图被用于产生门5和6（ 图2D）。这两个直方图的情节上的X轴和细胞计数在Y的轴的DNA含量s的定义的人口由3号门和4号门。直方图（ 图2E和2F）也可以被包含用于细胞的大小，如果需要的话（图中未示出）。对于细胞的大小的直方图，人口应设置门5和6，在X-轴和y-轴的计数绘制FSC。 </li><li>设置录音停止在10,000 GFP或RFP积极的事件（这将是4号门，派生门1号门，2号门和GFP或RFP积极的门）。范围从6,000 - 10,000 GFP阳性事件历来出版优质型材9,11的目标。我们建议使用最小的15翅膀或眼睛，约50％的GFP阳性获得足够的高品质型材10 4门事件。然而，我们已经成功地获得清晰的数据进行初步分析细胞周期从6翅膀（ 图3A）。设置采集量为300微升，这将使用总数的0.5毫升样品约460微升。 大表2显示了模板中所提供的例子在图2中的合适的阈值电压设置。 </li><li>运行样品在标准灵敏度或高灵敏度100微升/分钟和记录。我们发现几乎没有在我们的分析中差异与不同的灵敏度水平。由于的声聚焦细胞，流式细胞仪可以准确地测量样品在非常高的流速。不同于传统的流式细胞仪，我们发现没有数据通过收购速度高达每秒1500事件妥协。 Attune的软件所产生的数据文件记录。FCS格式，这是与大多数细胞周期建模软件兼容。 </li></ol> 5。数据分析<ol><li>绿色荧光蛋白（RFP）的细胞大小和DNA含量的直方图可以比较正面和负面的人群。他们可以手动分层，通过复制和粘贴使用Adobe Photoshop软件。如果在y轴被设定为自动，则在Y轴扩展的h每个样品计数ighest。叠加两个图的Y轴设置为自动定标，建立一个覆盖每个种群的全局最大值设置为与轴的直方图。这使得很容易在细胞周期的相位的相对变化的视觉比较，即使当GFP阳性细胞总数和GFP阴性群体是不等价的（例如，在图3B中）。另外，自定义y轴（y轴手动选项）可以被设定为一个固定值的直方图。在这种情况下，对两个群体的相对细胞数进行比较。由于上调样品每运行使用定义的体积，绝对量化为每个运行的细胞，在每个门与总人口的百分比，可以得到自动生成的统计信息表在运行过程中在工作区中（例如在图3F ）。点图和柱状图，也可以很容易地在分层Attune的软件，通过简单的拖放不同的样品从日E样品文件菜单右侧一个开放的样品到所需的图形进行比较。然而，覆盖亚群内单个样品（ 即 GFP阳性和GFP在同一样品阴性），我们使用Photoshop。 </li><li>两种方法都可以用来寻找人口的细胞周期的不同阶段的相对百分比。近似​​方法依赖于用户定义的区域划定边界的G1，S，G2和G2峰&gt;。这种方法允许快速和容易的百分比估计用户定义的区域内（ 图3E）。我们设定的区域，根据直接测试与果蝇 KC细胞S期，标记使用乙炔基-脱氧尿苷（EDU）注册成立。第二种方法是使用第三方建模软件，细胞周期分布的估计。例如，在图3F我们使用ModFitLT的软件（Verity的软件楼）。 </li></ol>

<ol>
	<li>Nunez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+measurement+and+cell+cycle+analysis+by+flow+cytometry.">DNA measurement and cell cycle analysis by flow cytometry.</a> Curr. Issues Mol. Biol. 3, 67-70 (2001).</li><li>Rabinovitch, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content+histogram+and+cell-cycle+analysis.">DNA content histogram and cell-cycle analysis.</a> Methods Cell Biol. 41, 263-296 (1994).</li><li>Ashburner, M., Roote, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila:+the+laboratory+setup.">Culture of Drosophila: the laboratory setup.</a> CSH Protoc. , pdb ip34 (2007).</li><li>del Valle Rodriguez, A., Didiano, D., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power+tools+for+gene+expression+and+clonal+analysis+in+Drosophila.">Power tools for gene expression and clonal analysis in Drosophila.</a> Nat. Methods. 9, 47-55 (2012).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34, 1-15 (2002).</li><li>McGuire, S. E., Mao, Z., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+gene+expression+targeting+with+the+TARGET+and+gene-switch+systems+in+Drosophila.">Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila.</a> Sci STKE. 2004, pl6 (2004).</li><li>Calvi, B. R., Lilly, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+BrdU+labeling+and+nuclear+flow+sorting+of+the+Drosophila+ovary.">Fluorescent BrdU labeling and nuclear flow sorting of the Drosophila ovary.</a> Methods Mol. Biol. 247, 203-213 (2004).</li><li>Bjorklund, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pathways+regulating+cell+size+and+cell-cycle+progression+by+RNAi.">Identification of pathways regulating cell size and cell-cycle progression by RNAi.</a> Nature. 439, 1009-1013 (2006).</li><li>Neufeld, T. P., de la Cruz, A. F., Johnston, L. A., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+growth+and+cell+division+in+the+Drosophila+wing.">Coordination of growth and cell division in the Drosophila wing.</a> Cell. 93, 1183-1193 (1998).</li><li>de la Cruz, A. F., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+Drosophila+cells.">Flow cytometric analysis of Drosophila cells.</a> Methods Mol. Biol. 420, 373-389 (2008).</li><li>Reis, T., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+regulation+of+dE2F1+by+cyclin-dependent+kinases+controls+cell+cycle+timing.">Negative regulation of dE2F1 by cyclin-dependent kinases controls cell cycle timing.</a> Cell. 117, 253-264 (2004).</li><li>Milan, M., Campuzano, S., Garcia-Bellido, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycling+and+patterned+cell+proliferation+in+the+Drosophila+wing+during+metamorphosis.">Cell cycling and patterned cell proliferation in the Drosophila wing during metamorphosis.</a> Proc. Natl. Acad. Sci. U. S. A. 93, 11687-11692 (1996).</li><li>Schubiger, M., Palka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+spatial+patterns+of+DNA+replication+in+the+developing+wing+of+Drosophila.">Changing spatial patterns of DNA replication in the developing wing of Drosophila.</a> Dev. Biol. 123, 145-153 (1987).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila; A laboratory handbook. , (1989).</li><li>Theodosiou, N. A., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+FLP/FRT+system+to+study+Drosophila+development.">Use of FLP/FRT system to study Drosophila development.</a> Methods. 14, 355-365 (1998).</li><li>Freeman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reiterative+use+of+the+EGF+receptor+triggers+differentiation+of+all+cell+types+in+the+Drosophila+eye.">Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye.</a> Cell. 87, 651-660 (1996).</li><li>Su, T. T., Sprenger, F., DiGregorio, P. J., Campbell, S. D., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P.H+Exit+from+mitosis+in+Drosophila+syncytial+embryos+requires+proteolysis+and+cyclin+degradation,+and+is+associated+with+localized+dephosphorylation.">P.H Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation.</a> Genes. Dev. 12, 1495-1503 (1998).</li></ol>

什么都没有透露。

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 ...

live-cell-cycle-analysis-drosophila-tissues-using-attune-acoustic

Research

JoVE Journal

Biology

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

13.6K 次观看.  University of Michigan. 活细胞周期分析的一种协议果蝇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在体内。

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

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

科研

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

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, ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子发生的细胞学分析:Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

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

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

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