特别感谢克里斯艺和贾森德尔里奥在这个项目的支持和援助。这项工作被授予GM070676石药RO1的支持。

苍蝇可以购买，并在实验室中扩增菌株。根据对果蝇文化收到小瓶量，需要频繁地“翻转”文化小瓶，小瓶已达到最大容量。扩增继续填充，直到大约50 果蝇的培养瓶。然后，一个“飞杯”三角帽100毫升烧杯（飞杯决策将在下一节讨论）。 24小时后，使用胚胎从这些杯种子新果 ​​蝇的培养瓶，贴在底部飞食品。在室温下从隔夜胚胎全面增长的苍蝇， 果蝇胚胎的生长时间约8.5天。种子的胚胎孵化到幼虫阶段后12-15小时。幼虫成长为4天左右蜕皮两次在24小时和48小时孵化后，进入第二和第三的幼虫阶段。幼虫，然后封装成pupariums和提交了4天的蜕变，直到新兴从T继承人蛹1例。大约两到三天的增长，在果蝇的培养瓶，允许在每一个苍蝇的数量增加。 400瓶充满时，会有足够的苍蝇，金额为50产蛋杯（每杯约1,000雌蝇）。苍蝇需要时间来适应新的环境。它通常需要大约两天为他们准备收集后，被转移到杯子。每天更换平板将保持干净的飞杯，这让苍蝇活得更长。 （ 果蝇的关怀和放大的进一步了解，请参考DB罗伯茨的果蝇 ：一个实用的方法2）。 一个很好的来源，获得大量的果蝇胚胎人口笼3，这是市售。人口笼的组装和维护，可以看出，在第5和第7章在书中果蝇 ：Practical利用细胞和分子生物学4。然而，人口笼是昂贵的，是很难维持的。由于人口的笼子可容纳丰富的苍蝇数量，使用二氧化碳转移和喂养苍蝇是必要的。如前所述，二氧化碳减少苍蝇的健康，使他们没有打好，以及。对规模小，100毫升三角帽烧杯都可以使用。 50 100毫升烧杯中，可能会达到9克dechorionated胚胎。为了消除死苍蝇，未使用的酵母膏和其它杂质，我们已经创建了一个不同的网目尺寸的双分层筛捕手。一个筛陷阱不必要的材料，如死苍蝇，同时允许通过胚胎（350微米孔径）。第二部分包含一个网格（孔径120微米），这是为了赶上果蝇胚胎和其他杂质，将通过网格。 1。飞杯的制备及胚胎收集<ol><li>翻动从多个小瓶的扩增到一个空塑料瓶的苍蝇，直到苍蝇的数量已达到一英寸瓶的高度。那么，那些苍蝇转移到100毫升鸡蛋收集杯（TRICON烧杯用尼龙网窗口）。在坚硬的表面保持攻杯转移苍蝇逃生时防止苍蝇。盖杯琼脂平板含有酵母膏，并允许稳定时间24-48小时。 </li><li>收集从五十飞杯隔夜的琼脂板和筛除尘器，使用干净的油漆刷和自来水洗板块的内容。肉用大量的水筛胚胎冲走，直到所有的酵母膏。 </li><li> Dechorionate在50％的漂白粉浸泡3分钟清洗的胚胎。 </li><li>广泛用蒸馏水冲洗，直到胚胎宽松的漂白剂气味。然后放置在AC折叠毛巾多次与胚胎干网。胚胎转移到一个干净的小瓶和记录重量。 &lt;/ LI&gt; </ol> 2。胚胎的同质化和澄清<ol><li>放置在Dounce均质与冰冷的提取液1.5 X卷dechorionated胚胎。 </li><li>做宽松杵5招。干净的离心管分装成匀浆。 15,000Ğ离心40分钟。在4°C </li><li>精心收集没有明确上清液上的白色脂肪层或较低的颗粒。 </li><li>将上清转移到清洁离心管，离心30分钟50,000Ğ。在4°C </li><li>由此产生的高速上清收集正如上文解释，可以立即使用，或可以捕捉冻结几个月储存于-80°C。 </li></ol> 3。微管聚合和驱动蛋白结合<ol><li>室温解冻的冷冻高速上清。聚合微管，添加的GTP（0.3毫米）和紫杉醇（20μM），并在室温下轻轻搅动20分钟旋转振动筛。 </li><li> kinesin的绑定，添加2.5毫米nonhydrolyzable ATP的模拟5&#39;-腺苷酸imidodiphosphate（AMPPNP），在室温10分钟旋转摇床搅拌。 </li></ol> 4。微管和驱动蛋白的不均匀沉降<ol><li>泥沙通过等量蔗糖垫在23,000克（sw41ti转子时，TLS-55）（20％的蔗糖和10μM的紫杉醇提取液），离心30分钟。在4°C </li><li>洗颗粒悬浮在原始提取缓冲区含有10μM的紫杉醇和75毫米氯化钠匀浆量的10％。 </li><li>重复步骤4.1与其他同等体积蔗糖垫沉淀。 </li><li>重新暂停5％提取液中的颗粒盐洗20微米紫杉醇，75毫米氯化钠， 硫酸镁 10毫米，和10毫米的三磷酸腺苷。 </li><li>泥沙在120,000克（sw41ti转子：31000转，TLS-55：42,000 RPM）为20分钟，4°C。收集上清（kinesin的分数）。 </li><li>执行14,000Ğ15分钟离心过滤，在4°C。恢复浓缩样品和30分钟重复了上述新的过滤器过滤和收集浓缩样本。 </li><li>进行蛋白质检测，以确定浓度。简单地说，不同的已知蛋白浓度（牛血清白蛋白BSA）混合与布拉德福德试剂的体积已知，在波长595nm处测定光密度。然后成立光密度与浓度的标准曲线计算kinesin的分数，此示例使用类似条件下的光密度测量标准牛血清白蛋白样品纯化的未知浓度。凝胶电泳以及免疫印迹也用纯化kinesin的金额。 </li><li>分装成小容量所需的浓度和在液氮中的单元冻结kinesin的分数存放在-80°C。 </li></ol>全长功能驱动蛋白纯化果蝇胚胎。 图1描述了银染凝胶，在纯化过程中的不同部位。巷1是高速上清澄清后的样品（步骤2.5），2巷澄清是一个沉淀的样品后，3车道是上清样品后第一次沉淀与蔗糖垫（步骤4.1），4车道是一个样品的颗粒先沉淀后，5巷是一个样品后的第二次沉淀（步骤4.3）上清，6车道是一个沉淀的样品后的第二次沉淀，7巷是样品颗粒最后沉淀（步骤4.5），8车道是kinesin的样品纯化，9巷过滤kinesin的样品，10巷是标记。集中在8日和9车道约115 kD的带动蛋白重链1，是与Figur一致萨克斯顿纸公布的1988年5巷8 E 1， 图2表示西部的c纯化kinesin的分数印迹检测使用动蛋白重链抗体。抗体AKINO1-A并没有显着交叉反应与其他动蛋白家族成员6。请注意，虽然此协议的结果，在功能动蛋白的良好来源，而分数动蛋白-1丰富，有可能的污染物，包括潜在的其他动蛋白和其他机动蛋白。我们删除，过滤各种较小的污染物。尽量消除它可以不被按尺寸做单独其它电机，净化类似什么，别人做研究驱动蛋白7，和我们相信，大多数活跃的电机是驱动蛋白-1，但更复杂的净化将允许潜力的汽车污染物的去除，如科尔等 8和萨克斯顿等 。 </p&gt; kinesin的processivity 在体外单分子微管结合实验Scholey题为“马达蛋白的运动功能检测”10书更详细的描述，评价。简单地说，用一个积极的电机的聚苯乙烯珠被带进接触与微管，在饱和的存在（1毫米）ATP。电机连接到微管，并开始步行激光陷阱中心。在预定义的珠陷阱中心的位移（100 nm）的激光束功率自动关闭，使电机空载下步行沿MT。影片展示了一个直径为500纳米的单kinesin的步行沿吨珠。视频画面的长度相当于20微米。 图3表示运行长度的测量单全长果蝇动蛋白分子的分布，纯化按照这里介绍的协议。在exponenti人适合的分布，提供游程平均单kinesin的1.55±0.1微米和1.28±0.12微米的过滤和过滤的样品分别。 <img src="/files/ftp_upload/3501/3501fig1.jpg" alt="图1" /> 图1纯化组分银染凝胶：从每个部分样品运行在10％的凝胶。被装在每个泳道10μg蛋白。巷1是高速上清澄清后的样品（步骤2.5），2巷澄清是一个沉淀的样品后，3车道是上清样品后第一次沉淀与蔗糖垫（步骤4.1），4车道是一个样品的颗粒先沉淀后，5巷是一个样品后的第二次沉淀（步骤4.3）上清，6车道是一个沉淀的样品后的第二次沉淀，7巷是样品颗粒最后沉淀（步骤4.5），8车道动蛋白纯化样品9巷是过滤kinesin的使用1 100 kD的截止Amicon超0.5毫升离心过滤（Millipore公司，美国）。蓝色箭头表示的蛋白质，其金额分别下降，红色箭头表示动蛋白的浓度，由于在该协议中使用的离心过滤步骤。 <img src="/files/ftp_upload/3501/3501fig2.jpg" alt="图2" /> 图2纯化餐旅学院分数抹杀反对反动蛋白抗体纯化kinesin的样品凝胶电泳和抹杀对初级抗体（硝酸纤维素膜）AKINO1一个1小时（TBST 1:1,000）在室温下孵育驴抗兔抗体（在TBST 1:10,000）在室温下一个小时。一个ECL试剂盒（化学发光）用于检测上面显示的kinesin的信号。 <img src="/files/ftp_upload/3501/3501fig3.jpg" alt="图3" /> 图3。 </strong&gt;游程和单一果蝇全长kinesin的速度：（一）及（B）过滤驱动蛋白样的游程和速度。 （三）及（D）游程和kinesin的样品过滤的速度。 图4。kinesin的运动视频。 <a href="http://www.jove.com/files/ftp_upload/3501/3501movie4.avi" target="_blank">点击这里观看视频</a> 。

<ol>
	<li>Ashburner, M., Thompson, J. N., Ashburner, M., Wright, T. R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+culture+of+Drosophila+2A.">The laboratory culture of Drosophila 2A.</a> The genetics and biology of Drosophila. , 1-81 (1978).</li><li>Roberts, D. B. <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 Practical Approach. , (1998).</li><li>Kunert, N., Brehm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+Drosophila+Embryosand+Chromatographic+Purification+of+Native+Protein+Complexes.">Mass production of Drosophila Embryosand Chromatographic Purification of Native Protein Complexes.</a> Methods in Molecular Biology. 420, 359 (2008).</li><li>Goldstein, L. S. B., Fyrberg, E. A., Wilson, L., Matsudaira, P. T., Eds, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+Practical+Uses+in+Cell+and+Molecular+Biology.">Drosophila melanogaster: Practical Uses in Cell and Molecular Biology.</a> Methods in Cell Biology. 44, (1994).</li><li>Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+kinesin:+Characterization+of+microtubule+motility+and+ATPase.">Drosophila kinesin: Characterization of microtubule motility and ATPase.</a> Proceedings of the National Academy of Science. 85, 1109 (1988).</li><li>Shubeita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1098 (2008).</li><li>Svoboda, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+Velocity+Measured+for+Single+Kinesin+Molecules.">Force and Velocity Measured for Single Kinesin Molecules.</a> Cell. 77, 773 (1994).</li><li>Cole, D. G., Saxton, W. M., Sheehan, K. B., Scholey, J. 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+"Slow"+Homotetrameric+Kinesin-related+Motor+Protein+Purified+from+Drosophila+embryos.">A "Slow" Homotetrameric Kinesin-related Motor Protein Purified from Drosophila embryos.</a> J. Biol. Chem. 269, 22917-22917 (1994).</li><li>Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Analysis+of+Microtuble+Motor+Proteins.">Isolation and Analysis of Microtuble Motor Proteins.</a> Drosophila Melanogaster. Bloomington: Academic. 44, 279 (1994).</li><li>Scholey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+Assay+for+Motor+Proteins.">Motility Assay for Motor Proteins.</a> Methods in Cell Biology. 39, 138 (1993).</li><li>Wagner, M. C., Pfister, K., Brody, S., Bloom, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+kinesin+from+bovine+brain+and+assay+of+microtubule-stimulated+ATPase+activity.">Purification of kinesin from bovine brain and assay of microtubule-stimulated ATPase activity.</a> Methods in Enzymology. 196, 157 (1991).</li><li>Aizawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+Family+in+Murine+Nerwous+System.">Kinesin Family in Murine Nerwous System.</a> The Journal of Cell Biology. 119, 1287-1287 (1992).</li><li>Ori-McKenney, K. M., Xu, J., Gross, S. P., Vallee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytoplasmic+dynein+tail+mutation+impairs+motor+processivity.">A cytoplasmic dynein tail mutation impairs motor processivity.</a> Nature Cell Biology. 12, 1228-1228 (2010).</li></ol>

我们什么都没有透露。

牛脑是最广泛使用的起始原料11净化全长kinesin的，虽然小鼠的大脑已被使用，以及12。使用牛脑动蛋白源作为一个大的缺点是新鲜的起始原料的可用性：屠宰场通常无法进入，必须非常新鲜，以取得积极的kinesin的大脑。此外，青年奶牛的大脑是有效的。最后，牛的基因操纵，目前没有一个可行的选择，所以只有野生型的蛋白质进行研究。 小鼠更容易，但收获?...

<table border="1"><tbody><tr><td> 试剂或材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>琼脂（颗粒） </td><td>费舍尔</td><td> 1423-500 </td><td></td></tr><tr><td>葡萄糖（D-葡萄糖），无水</td><td>费舍尔</td><td> D16-3 </td><td></td></tr><tr><td>培养皿</td><td>流式细胞</td><td> 35 1007 </td><td> 60x15mm风格 （聚酯）20/bag“ </td></tr><tr><td> 果蝇的培养瓶</td><td>国际自行车联盟</td><td></td><td></td></tr><tr><td>的Tricorn烧杯</td><td> Econo实验室公司的</td><td> B700-100 </td><td>体积：100毫升， 尺寸：58x72mm </td></tr><tr><td>酵母</td><td>红星</td><td> 2751 </td><td></td></tr><tr><td>尼龙网</td><td> genesse科学</td><td> 57-102 </td><td> 120米icron孔大小</td></tr><tr><td>尼龙网</td><td>小零件</td><td> 06-350/35 </td><td></td></tr><tr><td>管道</td><td>西格玛</td><td> 108321-27-3 </td><td></td></tr><tr><td>管道</td><td>西格玛</td><td> 108321-27-3 </td><td></td></tr><tr><td>甘油</td><td>费舍尔</td><td> 56-81-5 </td><td></td></tr><tr><td> EGTA </td><td>西格玛</td><td> 67-42-5 </td><td></td></tr><tr><td> MgS0 4（无水） </td><td>费舍尔</td><td> 7487-88-9 </td><td></td></tr><tr><td> PMSF </td><td>西格玛</td><td> 329-98-6 </td><td></td></tr><tr><td>亮肽素</td><td>西格玛</td><td> <a href="http://www.sigmaaldrich.com/catalog/Lookup.do?N5=CAS+No.&amp;N3=mode+matchpartialmax&amp;N4=103476-89-7&amp;D7=0&amp;D10=&amp;N25=0&amp;N1=S_ID&amp;ST=RS&amp;F=PR">103476-89-7</a> </td><td></td></tr><tr><td> aprotonin </td><td>西格玛</td><td> <a href="http://www.sigmaaldrich.com/catalog/Lookup.do?N5=CAS+No.&amp;N3=mode+matchpartialmax&amp;N4=9087-70-1&amp;D7=0&amp;D10=&amp;N25=0&amp;N1=S_ID&amp;ST=RS&amp;F=PR">9087-70-1</a> </td><td></td></tr><tr><td>驯服</td><td>西格玛</td><td> 178403-8 </td><td></td></tr><tr><td>性病</td><td> Calbiochem公司</td><td> 65635 </td><td></td></tr><tr><td> GTP的</td><td>西格玛</td><td> <a href="http://www.sigmaaldrich.com/catalog/Lookup.do?N5=CAS+No.&amp;N3=mode+matchpartialmax&amp;N4=36051-31-7&amp;D7=0&amp;D10=&amp;N25=0&amp;N1=S_ID&amp;ST=RS&amp;F=PR">36051-31-7</a> </td><td></td></tr><tr><td>紫杉醇</td><td>西格玛</td><td> 33069-62-4 </td><td></td></tr><tr><td> ATP类似物5&#39;-腺苷酸imidodiphosphate </td><td>西格玛</td><td> 3605-31-7 </td><td></td></tr><tr><td>氯化钠</td><td> mallinckrodt </td><td> 7647-14-5 </td><td></td></tr><tr><td>三磷酸腺苷</td><td>西格玛</td><td> 74804-12-9 </td><td></td></tr><tr><td> amicon超离心过滤器.5毫升，100KD切断，8折</td><td> Millipore公司</td><td> UFC510008 </td><td></td></tr></tbody></table>

isolation and purification of kinesin from drosophila embryos

马达蛋白沿着微管移动的货物，并运送到特定的亚细胞位置。由于改变交通建议多种神经退行性疾病的基础，了解微管的汽车运输及其调控最终可能会导致更好的治疗方法。动蛋白是真核细胞马达蛋白在ATP水解供电，沿微管（MTS）顺（加末）的方向移动。在这里，我们报告了详细的净化从果蝇胚胎中分离的积极全长kinesin的协议，从而使单分子生物物理研究果蝇遗传学相结合。拥有大约1000名女性，每杯，约50铺设杯开始，我们进行了隔夜的集合。这提供了约10毫升装的胚胎。胚胎漂白dechorionated（产生的胚胎约9克），然后均匀。 AF之三的破坏，匀浆，利用低速旋转，由高速离心澄清。澄清的上清液治疗GTP和紫杉醇微管聚合。动蛋白聚合微管上，通过增加ATP的模拟在室温下，5&#39;-腺苷酸imidodiphosphate固定。 kinesin的绑定后，微管沉降的通过通过蔗糖坐垫高速离心。微球，然后再暂停，并重复此过程。最后，ATP的释放从MTS动蛋白。高速离心，然后降速MTS，留在上清动蛋白。这kinesin的使用100第纳尔切断进一步净化过滤离心过滤，分装，捕捉液氮冷冻，储存在-80°C。使用纯化的样品进行SDS凝胶电泳和免疫印迹。最后离心过滤步骤之前和之后的纯化的样品电机活动在体外单分子微管法进行了评估。离心过滤前后动蛋白组分显示processivity以前文献报道。进一步的实验正在进行评估动蛋白和其他运输相关的蛋白质之间的相互作用。

Watch this Scientific Journal Video about Isolation and Purification of Kinesin from Drosophila Embryos at JoVE.com

Isolation and Purification of Kinesin from Drosophila Embryos

这是一个协议隔离积极全长驱动蛋白<em&gt;果蝇</em&gt;单分子生物物理研究的胚胎。我们如何收集胚胎，使胚胎裂解，然后聚合微管（MTS）。动蛋白纯化固定在MTS，纺纱动蛋白吨复合物，然后释放从MTS动蛋白通过磷酸腺苷此外。

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 ...

isolation-and-purification-of-kinesin-from-drosophila-embryos

Research

JoVE Journal

Biology

Isolation and Purification of Kinesin from Drosophila Embryos

12.1K 次观看.  University of California, Irvine. 这是一个协议隔离积极全长驱动蛋白果蝇单分子生物物理研究的胚胎。我们如何收集胚胎，使胚胎裂解，然后聚合微管（MTS）。动蛋白纯化固定在MTS，纺纱动蛋白吨复合物，然后释放从MTS动蛋白通过磷酸腺苷此外。

视频: Isolation and Purification of Kinesin from Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

科研

生物学

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

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 ...

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 ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

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. ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

TRAP-RC，翻译核糖体亲和纯化的稀有细胞群<em&gt;果蝇</em&gt;胚胎

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...