作者感谢Veraksa实验室的成员对手稿的帮助意见和协议改进建议。 AV由NIH授权GM105813和NS096402支持。 LY得到马萨诸塞州大学波士顿赛诺菲药学博士学位的支持。

5.制备5L飞行笼（适合15cm板） <ol><li>获得所需材料:带盖的5夸脱（4.7升）容器（ 图1A ），尼龙网和剃刀刀片。 </li><li>标记一个12厘米直径的圆圈，并使用剃刀刀片在容器底部切开一个孔（ 图1B ）。在盖子上切割一个15厘米直径的孔（ 图1C ）。 </li><li>将尼龙网切成25×25厘米的正方形。 </li><li>将网格放在容器的顶部边缘上，然后用盖子向下按压（ 图1C ）。确保网格紧固，盖子不会倾斜。笼子已准备好安装苍蝇（参见步骤5.3）。 </li></ol> 2.制备1L飞行笼（适合10cm板） <ol><li>获得所需材料:电钻，3英寸（76毫米）切割工具（ 图1E和F）图1F​​），带盖的直立1L塑料罐，尼龙网，剃刀刀片，砂纸，护目镜和工作手套。 </li><li>在底部和塑料瓶的盖子上切下3英寸（76毫米）的孔（ 图1G ），盖子在钻孔时紧紧地拧入容器。开始制作一个孔时，将切削刀具定位在圆心的中央。 注意:在此步骤中佩戴眼睛防护和工作手套。 </li><li>用剃刀刀片和砂纸平滑孔的边缘。 </li><li>将尼龙网切成18×18厘米的正方形。 </li><li>将网格放在瓶子的顶部边缘上并拧紧盖子（ 图1G ）。确保网格紧固，盖子不会倾斜。笼子已准备好安装苍蝇（参见步骤5.3）。 </li></ol> 3.苹果汁（AJ）板的制备<ol><li>将19g琼脂和1L18.2MΩ·cm的水加入2L烧瓶中。添加搅拌棒，并简单混合。 </li><li>高压灭菌30分钟同时，从冷冻原料中解冻100％苹果汁浓缩物，并制备10mL 10％（重量/体积）4-羟基苯甲酸甲酯溶液在乙醇中。 </li><li>高压灭菌后，开始将搅拌板上的琼脂混合，加入80 mL 100％苹果汁浓缩液和10mL 10％（重量/体积）的4-羟基苯甲酸甲酯溶液至烧瓶中。在室温下保持恒温混合。 </li><li>倒入15厘米培养皿（5升笼子）或10厘米培养皿（1升笼子），以使每个培养皿中的琼脂水平约为5毫米厚。允许在室温下固化，并包装在塑料袋中。 AJ板可以在4℃下储存两周。 注意:培养皿可以洗涤和重复使用。 </li><li>准备湿酵母糊:将一些活性干酵母倒入具有容易混合的盖子的100mL容器中，并在搅拌下少量加水，得到浓稠但充分混合糊。在4°C下储存长达2周。 </li></ol> 4.裂解试剂的制备<ol><li> 制备5x裂解缓冲液:250mM Tris pH 7.5,25％甘油，1％辛基苯氧基聚（乙烯氧基）乙醇（IGEPAL CA-630），7.5mM MgCl 2，625mM NaCl，125mM NaF，5mM Na 3 VO 4 。 <ol><li>制成200毫升:将1g NaF粉末和184毫克Na 3 VO 4粉末在不断搅拌下在71毫升水中完全溶解。加入50mL 1M Tris pH 7.5,2mL 100％IGEPAL，1.5mL 1M MgCl 2和25mL 5M NaCl。然后加入50mL甘油（最后加入），继续搅拌1小时。 </li><li>使用250 mL过滤器过滤灭菌。该步骤可能很慢，但过滤溶液对于去除不溶性颗粒很重要。在50mL管中分装10 mL，储存于-80°C。 </li></ol></li><li>在水中制备1 M二硫苏糖醇（DTT）溶液。在-20℃下以1mL等分试样储存。 </li><li>获得蛋白质ase抑制剂混合片，与乙二胺四乙酸（EDTA）。储存于4°C。 </li><li>获得10 mL注射器和注射器过滤器:直径26 mm，无表面活性剂的醋酸纤维素（SFCA）膜，孔径0.45μm。 </li></ol>飞鸟收集<ol><li>扩增携带有标签的蛋白质的转基因飞线（每瓶约100只苍蝇），每2天转运瓶子，直到每条飞行线累积25-30瓶。以类似的方式放大控制库存（ 例如 yw线）。 </li><li>温热AJ板至室温（约1-2小时，可以一夜之间完成）。使用戴手套的手指，将一些湿酵母膏撒在AJ板的中心， 6厘米圆形，15厘米板和4厘米圆形的10厘米板。 </li><li> 使用标准的果蝇阶段设置，用二氧化碳气流麻醉苍蝇，并转入笼子（每5L笼子大约15只成熟的苍蝇的瓶子，每1L笼子5瓶）。 <ol><li>用步骤5.2制备的AJ板覆盖笼底，并使用两条胶带将AJ板粘贴到笼上（ 图1D和1H ）。折叠板上的胶带的顶端，以便在更换印版时易于剥离是有用的。 </li><li>苍蝇从麻醉中恢复后，将笼子保持在底部，并在顶部网格。 注意:重要的是等到苍蝇完全恢复，否则它们会粘在AJ板和酵母酱上。 </li><li>在实验结束时，为了准备笼子进行清洁，将笼子放入冷藏室几个小时。将网状物浸泡在水中几个小时将有助于清洁。 </li></ol></li><li>孵化笼中的苍蝇在室温2-3天，并更换AJ板每天两次。要更换盘子，请将笼子倒置，从盘子上取出胶带，然后将盘子固定在笼子上，轻轻地将整个笼子轻轻地放在板凳上，快速地将旧板子换掉一个新的盘子，尽量不要让苍蝇逃跑。 注意:苍蝇需要适应笼子，最好从第3天开始。他们将在高峰期继续铺设约4-5天。两个5 L的笼养能力可以在过夜收集中产生高达1 g的胚胎。使用1 L笼子进行较小规模的实验。 </li><li>允许苍蝇将新鲜AJ板上的胚胎过夜（约16小时）或任何其他所需的时间长度。 </li><li>第二天早上，将每个飞行线用于每个飞行线，用于胚胎收集的标记和称重网格容器。 </li><li> 使用第4部分的试剂制备1x裂解缓冲液（注:即将使用的1x裂解缓冲液必须在提取前准备）:向10 mL 5x浓缩的裂解缓冲液（储存于-80°C）中加入40 mL水，在50mL管中。 <ol><li>加入50μL的1 MDTT至终浓度为1mM，充分混合并分成两个50mL管，每个管中25mL。向其中一个管中加入一种蛋白酶抑制剂片剂，并在4℃下旋转30分钟。当片剂溶解，收集和解冻胚胎（步骤5.8至6.6）。检查以确保平板电脑已完全溶解，并将缓冲液保持在冰上。 注意:带有DTT的1x缓冲液管可以保存在-80°C，只需要在下一次实验之前添加平板电脑。考虑到蛋白质纯化步骤中的后续洗涤，25mL裂解缓冲液对于多达2个样品是足够的。 </li></ol></li><li>将AJ板放在使用飞线的基因型的笼子上，并将盘更换为新鲜的。 注意:在此步骤中可以从板上刮掉过量的酵母膏。胚胎可以在25℃或室温下在板上老化，以获得期望的发育时间窗口。 </li><li> 添加足够的水to覆盖每个AJ板。用柔软的油漆刷轻轻地将胚胎从板上取下（平头约10毫米宽适用于大板）。将胚胎倒入以前称重和标记的网状容器中，并排出多余的水分。 <ol><li>如果需要，进行第二次清洗和刷涂板以除去所有的胚胎。将来自相同飞线的附加板的胚胎合并到相同的网状容器中。 </li></ol></li><li>简单地在纸巾上清除网眼以除去多余的水分。 </li></ol>胚胎脱层<ol><li>半满6厘米培养皿与50％漂白剂（体积/体积溶液在水中）。 注意:请参见材料和试剂表中关于漂白剂品牌的重要评论。 注意:戴上手套，避免在衣服上漂白。 </li><li>准备2个较大的培养皿用水。 </li><li>将含有胚胎的网状容器浸入50％漂白剂中90秒。在入口处轻轻点击网格在漂白剂溶液中暂停胚胎几次。 </li><li>在90s孵育结束时，用水洗涤每个2个皿中的网状容器约10s。 </li><li>用喷水瓶用18.2MΩ·cm的水冲洗网状容器。此外，洗涤网状容器的侧面和外部。 注意:彻底清洗后不应检测到漂白气味。 </li><li>将网状容器放在纸巾上。用胚胎称重网状容器，并减去步骤5.6中获得的空容器的重量。记录胚胎的最终重量。适量的胚胎是0.5 - 1 g。 </li></ol> 7.胚胎提取物的制备<ol><li>加入1 mL具有蛋白酶抑制剂的裂解缓冲液（从步骤5.7）至均匀玻璃匀浆器，并保持在冰上。将胚胎从网状容器转移到匀浆器中。 注意:胚胎可以用平头o拾取金属刮刀或刷子直接转移到缓冲器中。在冰面上执行本节中的所有步骤。 </li><li>用额外的1 mL裂解缓冲液洗涤网上剩余的胚胎，并加入匀浆器中的材料。让胚胎沉淀到匀浆器的底部，并小心吸出上清液。 </li><li>将蛋白酶抑制剂（来自步骤5.7）的裂解缓冲液加入匀浆器中，目标为每克胚胎5-6mL裂解缓冲液。 </li><li>用松散的杵将胚胎均匀化4-6次。 注意:在第一次冲击中，会有更多的阻力。尝试连续移动杵，以免溅出溶液。 </li><li>用紧密的杵将胚胎均匀化8-10次。 </li><li>将匀浆在冰上孵育20分钟。 </li><li>将匀浆转移到微量离心管中，并以最高速度（约13,000 xg）在4℃离心15分钟。 </li><li> Avoidi将颗粒和最上层脂质层转移到新鲜的微量离心管中，并以最高速度（约13,000xg）在4℃离心15分钟。 </li><li>仔细吸取1毫升尖端的上清液，并装入冷冻的10mL注射器中，并连接0.45μm过滤器（将相同样品的等分试样装入一个注射器）。将所有溶液推入冰上标记的15 mL管中。提取物可以立即用于随后的蛋白质纯化实验或在液氮中快速冷冻，并保持在-80℃以备将来使用。 注意:最好将提取物而不是胚胎冷冻，因为在随后的解冻过程中，蛋白酶可能会被激活并导致蛋白质降解。 </li></ol>

<ol>
	<li>Veraksa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+developmental+processes:+insights+from+mass+spectrometry-based+proteomics.">Regulation of developmental processes: insights from mass spectrometry-based proteomics.</a> Wiley Interdiscip Rev Dev Biol. 2 (5), 723-734 (2013).</li><li>Gavin, A. C., Maeda, K., Kuhner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+charting+protein-protein+interaction:+mass+spectrometry-based+approaches.">Recent advances in charting protein-protein interaction: mass spectrometry-based approaches.</a> Curr Opin Biotechnol. 22 (1), 42-49 (2011).</li><li>Arbeitman, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+during+the+life+cycle+of+Drosophila+melanogaster.">Gene expression during the life cycle of Drosophila melanogaster.</a> Science. 297 (5590), 2270-2275 (2002).</li><li>Soeller, W. C., Poole, S. J., Kornberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transcription+of+the+Drosophila+engrailed+gene.">In vitro transcription of the Drosophila engrailed gene.</a> Genes Dev. 2 (1), 68-81 (1988).</li><li>Kamakaka, R. T., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soluble+nuclear+fraction,+a+highly+efficient+transcription+extract+from+Drosophila+embryos.">The soluble nuclear fraction, a highly efficient transcription extract from Drosophila embryos.</a> Methods Cell Biol. 44, 225-235 (1994).</li><li>Kyriakakis, P., Tipping, M., Abed, L., Veraksa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+in+Drosophila:+The+advantages+of+the+GS-TAP+system.">Tandem affinity purification in Drosophila: The advantages of the GS-TAP system.</a> Fly (Austin). 2 (4), 229-235 (2008).</li><li>Shaffer, C. D., Wuller, J. M., Elgin, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raising+large+quantities+of+Drosophila+for+biochemical+experiments.">Raising large quantities of Drosophila for biochemical experiments.</a> Methods Cell Biol. 44, 99-108 (1994).</li><li>Sisson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+large+populations+of+Drosophila+for+protein+biochemistry.">Culturing large populations of Drosophila for protein biochemistry.</a> CSH Protoc. 2007, (2007).</li><li>Caravaca, J. M., Lei, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+a+Drosophila+melanogaster+Population+Cage.">Maintenance of a Drosophila melanogaster Population Cage.</a> J Vis Exp. (109), (2016).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minibrain+and+Wings+apart+control+organ+growth+and+tissue+patterning+through+down-regulation+of+Capicua.">Minibrain and Wings apart control organ growth and tissue patterning through down-regulation of Capicua.</a> Proc Natl Acad Sci U S A. 113 (38), 10583-10588 (2016).</li><li>Neumuller, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stringent+analysis+of+gene+function+and+protein-protein+interactions+using+fluorescently+tagged+genes.">Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes.</a> Genetics. 190 (3), 931-940 (2012).</li><li>Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+complexes+in+Drosophila+with+tandem+affinity+purification-mass+spectrometry.">Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.</a> Dev Dyn. 232 (3), 827-834 (2005).</li><li>Mukherjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Notch+signalling+by+non-visual+beta-arrestin.">Regulation of Notch signalling by non-visual beta-arrestin.</a> Nat Cell Biol. 7 (12), 1191-1201 (2005).</li><li>Gilbert, M. M., Tipping, M., Veraksa, A., Moberg, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screen+for+Conditional+Growth+Suppressor+Genes+Identifies+the+Drosophila+Homolog+of+HD-PTP+as+a+Regulator+of+the+Oncoprotein+Yorkie.">A Screen for Conditional Growth Suppressor Genes Identifies the Drosophila Homolog of HD-PTP as a Regulator of the Oncoprotein Yorkie.</a> Dev Cell. 20 (5), 700-712 (2011).</li><li>Anjum, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Toll+Signaling+and+Inflammation+by+beta-Arrestin+and+the+SUMO+Protease+Ulp1.">Regulation of Toll Signaling and Inflammation by beta-Arrestin and the SUMO Protease Ulp1.</a> Genetics. 195 (4), 1307-1317 (2013).</li><li>Degoutin, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riquiqui+and+minibrain+are+regulators+of+the+hippo+pathway+downstream+of+Dachsous.">Riquiqui and minibrain are regulators of the hippo pathway downstream of Dachsous.</a> Nat Cell Biol. 15 (10), 1176-1185 (2013).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecdysone+receptor+coactivator+Taiman+links+Yorkie+to+transcriptional+control+of+germline+stem+cell+factors+in+somatic+tissue.">The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue.</a> Dev Cell. 34 (2), 168-180 (2015).</li><li>Lemmon, M. A., Schlessinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+by+receptor+tyrosine+kinases.">Cell signaling by receptor tyrosine kinases.</a> Cell. 141 (7), 1117-1134 (2010).</li><li>Vincent, J. P., Girdham, C. H., 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=A+cell-autonomous,+ubiquitous+marker+for+the+analysis+of+Drosophila+genetic+mosaics.">A cell-autonomous, ubiquitous marker for the analysis of Drosophila genetic mosaics.</a> Dev Biol. 164 (1), 328-331 (1994).</li><li>Zhai, B., Villen, J., Beausoleil, S. A., Mintseris, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+Drosophila+melanogaster+embryos.">Phosphoproteome analysis of Drosophila melanogaster embryos.</a> J Proteome Res. 7 (4), 1675-1682 (2008).</li><li>Franco, M., Seyfried, N. T., Brand, A. H., Peng, J., Mayor, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+to+isolate+ubiquitin+conjugates+reveals+wide+role+for+ubiquitination+during+neural+development.">A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.</a> Mol Cell Proteomics. 10 (5), (2011).</li><li>Ramirez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+the+Ubiquitin+Landscape+in+the+Drosophila+Embryonic+Nervous+System+and+the+Adult+Photoreceptor+Cells.">Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells.</a> PLoS One. 10 (10), 0139083 (2015).</li></ol>

作者没有什么可以披露的。

这里提出的方案是一个简单和一般的程序，用于在中等规模设置果蝇种群笼子，并从胚胎中制备全细胞蛋白质提取物。所得提取物可用于各种下游应用，例如在亲和树脂上纯化蛋白质复合物。在冰上进行提取步骤和使用强烈的蛋白酶抑制是至关重要的，以最小化蛋白质降解。如所述，该方案适用于分离大多数细胞质和一些膜和可溶性核蛋白6,10,12,15。它可以容易地适应于来自其他隔室的更集...

遗传筛选和最近的基因组方法已经彻底改变了生物功能的研究。然而，重要的细胞信息被编码在蛋白质及其相互作用的合作伙伴的整体中。虽然传统的基因修饰屏可以识别速率限制途径成分并恢复间接相互作用，但蛋白质组学方法的强度在于它们识别感兴趣的蛋白质的完全即时相互作用网络的能力。因此，蛋白质组学是研究生物系统的有价值的正交方法，补充基因组学，转录组学和传统遗传筛选。亲和纯化 - 质谱（AP-MS）已被证明是研究细胞和组织中天然环境中蛋白质 - 蛋白质相互作用（PPI）的有力方法。这种方法允许在特定发展中识别直接或间接的相互作用已经成功地用于鉴定多种发育途径中的多种新型PPI（参考文献1 ）。尽管PPI研究取得了无可争议的成功，但其大部分已经在培养的细胞中进行，其中感兴趣的"诱饵"蛋白质被过度表达。在细胞培养中研究PPI有两个问题:首先，特定的细胞系由于缺乏某些蛋白质的表达而不能提供完整的相互作用。第二，通常在这种分析中使用的高过表达可能导致人为的物质，如蛋白质错误折叠或假阳性相互作用的识别。 这两个限制都可以通过在体内分析PPI来克服。这种实验中的限制性步骤是可用于纯化蛋白质复合物的原料。 黑腹果蝇长期以来一直被用作功能分析的模型sis，最近也被证明是在体内研究PPIs的优秀体系。 果蝇胚胎发生是研究PPI的特别有吸引力的组织类型，因为可以容易地收集胚胎，并且也因为大多数基因（&gt; 88％）在胚胎发生过程中被表达，因此提供了丰富的体内环境来检测相关的PPIs 3 。 传统上，苍蝇的生物化学研究利用非常大规模的胚胎收集（100-150 g），如纯化功能转录裂解物4,5所必需的。以前在果蝇中的 AP-MS研究也需要大量的胚胎（ 5-10g ），因为它们依赖于两步纯化方法，例如串联亲和纯化（TAP），每个步骤6都有相关的材料损失。大型的起始材料需要在大型人口笼中设置胚胎收集，这可能是昂贵的（在商业购买时）并且耗时地维持和清洁7,8,9 。 单步亲和纯化方法的发展的最新进展以及质谱仪的增加的灵敏度已将原料的必要量减少一个数量级。使用诸如链霉抗生物素蛋白结合肽（SBP）或绿色荧光蛋白（GFP）的标签并从小于1g的胚胎开始，可以分离足以鉴定的诱饵蛋白质和相互作用成分的量质谱法10,11 。 这里提出的协议的目标是帮助研究人员我感受到体内 PPI 的生物化学分析的障碍。为此，我们提供了一种简单而廉价的方法来收集中等规模（0.5-1 g）的果蝇胚胎，然后一步制备适合随后通过AP-MS或其他方法分析的全细胞蛋白质提取物。我们的方法依赖于使用可由任何实验室轻松生产的定制的1-L或5-L种群笼。此外，这里提出的提取条件已经在几个研究中验证，在培养的细胞中和在体内 10,12,13,14,15,16,17。

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Container (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209330</td>
			<td style="width:644px;">For making 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Lid (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209320</td>
			<td style="width:644px;">Diameter 8.37 in. Lids for 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 100 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 1-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 150 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875714</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 60 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td style="width:644px;">Material: Polystyrene. Used during embryo dechorionation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sefar NITEX nylon mesh</td>
			<td style="width:183px;">Sefar</td>
			<td style="width:119px;">03-180/44</td>
			<td style="width:644px;">180 micron, 44% open area, used for fly cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Milwaukee 3 in. Hole Dozer Hole Saw with Arbor</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">49-56-9670</td>
			<td style="width:644px;">3 inch cutting tool for making 1-L cages.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Wide-Mouth Straight-Sided PMP Jars with White Polypropylene Screw Closure</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">11-823-33</td>
			<td style="width:644px;">For making 1-L cages. Thermo Scientific cat # 21171000. Alternative 1-L containers can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Red Star Active Dry Yeast, 2 pound pouch</td>
			<td style="width:183px;">Red Star&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from Amazon.com or other suppliers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Frozen 100% Apple Juice Concentrate&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from a grocery store. Has to say &#34;100% apple juice&#34; on the can.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:183px;">Acros Organics</td>
			<td style="width:119px;">AC126965000</td>
			<td style="width:644px;">also known as methylparaben or Tegosept. Used as a preservative in AJ plates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">IGEPAL CA-630 for molecular biology, 100 ml</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">I8896</td>
			<td style="width:644px;">used for preparing lysis buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Rapid-Flow Sterile Disposable Filter Units with CN Membrane&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-740-2A</td>
			<td style="width:644px;">0.2 &#956;m pore size. Used for filtering 5x lysis buffer. Thermo Scientific cat # 1260020.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">CO-RO ROCHE cOmplete Protease Inhibitor Cocktail</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">11697498001</td>
			<td style="width:644px;">Vial of 20 tablets. Used for protease inhibition in lysis buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning SFCA syringe filters&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-754-21</td>
			<td style="width:644px;">SFCA membrane, diameter 26 mm, pore size 0.45 &#956;m. Used for filtering final extract samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">BD&#160;Luer-Lok Disposable Syringes without Needles</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">14-823-2A</td>
			<td style="width:644px;">for filtering final extract samples. BD cat # 309604.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:439px;">Bleach</td>
			<td style="width:183px;">Clorox</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">used for embryo dechorionation at 50% (vol/vol) in water, can be purchased at any supermarket. It is important to use the Clorox brand, as other brands may result in incomplete dechorionation or may be toxic for embryos.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning Costar Netwell Plates</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-213</td>
			<td style="width:644px;">mesh containers for embryo collections. 74 &#956;m mesh size, 6-well.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Wheaton Dounce Tissue Grinders, capacity 15 ml</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">06-435B</td>
			<td style="width:644px;">homogenizer for embryos, with loose and tight pestles. Wheaton cat # 357544.</td>
		</tr>
	</tbody>
</table>

medium-scale preparation of drosophila embryo extracts for proteomic experiments

蛋白质 - 蛋白质相互作用（PPI）的分析已经成为研究生物过程和机制，如细胞信号传导，生物发育和疾病的不可或缺的方法。通常希望使用体内材料获得PPI信息，以获得最自然和无偏见的互动网络视图。果蝇果蝇黑腹果蝇是体内研究PPIs的绝佳平台，适用于生物化学实验隔离材料的简单方法。特别地，由于在该发育阶段易于收集动物以及大多数蛋白质在胚胎发生中表达，所以果蝇胚代表研究PPI的便利类型的组织，从而提供了显示大多数PPI的相关环境。在这里，我们提出了中等规模收集果蝇胚胎的方案（0.5-1 g），这是一个广泛的蛋白质组学应用，包括通过亲和纯化 - 质谱（AP-MS）分析PPI。我们描述我们的设计为1 L和5 L网箱的胚胎收集，可以轻松，廉价地设置在任何实验室。我们还提供用于胚胎收集和蛋白质提取的通用方案，以产生可直接用于下游应用如AP-MS的裂解物。我们的目标是为所有研究人员提供一种可行的手段来进行体内 PPI的分析。

为了说明在蛋白质复合物纯化实验中使用该方案，我们在控制下产生了一种纯合可行的飞行线，表达EGFP标记的果蝇细胞外信号调节激酶（由滚动基因编码的ERK）的臂EGFP-ERK的普遍存在的犰狳 （ 臂 ）启动子18,19 。在果蝇胚胎发生的大多数阶段， 臂启动子是有活性的。使用所述方案制备裂解物，通过蛋白质印...

Watch this Scientific Journal Video about Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments at JoVE.com

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

该方案的目标是提供一种直接和廉价的方法来收集中等规模的果蝇胚胎（0.5-1 g），并制备可用于下游蛋白质组学应用的蛋白质提取物，如亲和纯化 - 质谱（AP-MS ）。

中等规模的制备<em&gt;果蝇</em&gt;蛋白质组学实验胚胎提取物

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

medium-scale-preparation-drosophila-embryo-extracts-for-proteomic

Research

JoVE Journal

Developmental Biology

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

7.8K 次观看.  University of Massachusetts Boston. 该方案的目标是提供一种直接和廉价的方法来收集中等规模的果蝇胚胎（0.5-1 g），并制备可用于下游蛋白质组学应用的蛋白质提取物，如亲和纯化 - 质谱（AP-MS ）。 

视频: Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

科研

发育生物学

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

在代谢分析准备线粒体富集级的<em&gt;果蝇</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

两鲑鱼鱼、虹鳟 (虹鳟鱼虹鳟鱼) 和褐鳟 (鲑鲑形态石斑鱼) 的空气剥离效果

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

无珠光高音喇叭在食子胚胎中的细胞力学探测

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...