多亏了印第安纳大学的大众光谱学设施和犹他大学新陈代谢的核心设施来帮助优化这个协议。J.M.T. 由国立卫生研究院国立医学研究所 R35GM119557 奖编号支持。

1. 收集卵子
<ol><li>收集所需基因型的成年男性和处女女性。单独年龄这些动物在一个食物小瓶与标准布卢明顿媒介为3–5天。</li>
	<li>通过将50只处女母和25只雄性转移到一个新的食品瓶中, 建立适当的交配。 注: 每种基因型应至少设置六个独立交配。每种交配 (即从六个独立的交配收集的六个样本) 中只收集一个样本。</li>
	<li>
准备糖蜜下蛋帽。
	<ol>
<li>将115毫升糖蜜和29克琼脂与700毫升 H2O 在 2 L 瓶中混合。</li>
		<li>在热盘子上煮沸糖蜜琼脂混合物。</li>
		<li>冷却到70摄氏度。</li>
		<li>添加25毫升的酸混合 (20 毫升85% 磷酸和209毫升丙酸在 1 L 的 H2O;贮存在棕色瓶) 和10毫升10% 对羟基苯甲酸甲酯在95% 乙醇。</li>
		<li>将糖蜜琼脂倒入 35 x 10 毫米2组织培养皿的盖子和下半部分。 注: 在浇注糖蜜琼脂板之前, 确保 35 x 10 mm2板的盖子和/或底座适合 snuggly 到果蝇股票瓶 (步骤1.6 中描述) 的口中。根据使用的车牌品牌, 底座可能无法使用。</li>
	</ol>
</li>
	<li>将蒸馏水添加到活性干酵母中 (5 克 yeast/7 毫升水), 直到混合物变成糊状, 可以很容易地扩散到固体表面 (即, 花生黄油的一致性)。</li>
	<li>在糖蜜下蛋帽的表面上散布约1.5 克的酵母膏。</li>
	<li>用22克针戳四个空气孔到塑料6盎司的反面.果蝇股票瓶。</li>
	<li>将新交配的苍蝇从食物瓶转移到培养瓶中。 注: 苍蝇可以通过使用 CO2作为临时麻醉剂或通过保持瓶子的顶部, 并尖锐地敲击瓶子底部的台式。</li>
	<li>快速地放置一个糖蜜蛋盖与酵母糊在表面到一个果蝇瓶的嘴里。使用实验室胶带, 以确保产卵帽到位。 注: 如果苍蝇在转移前被麻醉, 应将瓶子水平放置在台式上, 直到所有苍蝇都恢复正常为止。一旦苍蝇是完全移动, 把瓶子放在一个孵化器与糖蜜产卵帽的文化底部。</li>
	<li>在头两天至少更换一次糖蜜蛋帽, 先反转股票瓶, 敲击瓶子底部的台式, 去掉旧的蛋帽, 然后立即将新的蛋帽放进瓶子的嘴里。扔掉旧的下蛋帽。 注意: 这一步确保雌性不持有卵子, 并允许收集同步种群。</li>
	<li>在3天后, 将一个新的下蛋帽放入库存瓶中, 在2小时后更换, 然后丢弃。在四小时后取出并更换第二个下蛋帽。用日期、时间和基因型标记这个下蛋帽的底部。 注: 在这4小时的窗口收集的鸡蛋将用于分析。此同步步骤对于分析特定的开发阶段至关重要。鸡蛋可以用这种方式收集好几天。</li>
	<li>将产卵帽插入 60 x 15 毫米2 Petri 板, 并将其置于理想的温度和湿度的孵化器中。</li>
	<li>每天检查产卵帽中的饥饿、人口密度或污染问题。 注意: 幼虫必须获得足够的食物, 以确保持续发展。如有必要, 新鲜的酵母膏可以在所有产卵帽上传播, 这将在实验中使用。此外, 如果幼虫种群密度过高, 幼虫就会开始从培养盘中游走。这些样本不应用于 metabolomic 分析, 因为高人口密度和中间爆发的饥饿经历, 而徘徊将导致数据不一致。建议每片80–100中二龄幼虫 (L2) 的密度为每片或40–50中第三龄幼虫 (L3)。应丢弃明显受细菌或真菌污染的样品。</li>
</ol>2. 幼虫标本采集
<ol><li>年龄幼虫直到理想的阶段。如果收集 L3 幼虫, 重新同步样品在 L2–L3 蜕皮。通过使用前面的气孔作为开发里程碑15的以前描述的方法, 通常实现重新同步。或者, mid-L3 幼虫可以使用sgs3报告基因16进行同步。 注: 在这方面, 我们发现 mid-L2 幼虫 (产卵后60小时) 最适合于大多数分析, 因为幼虫发育在这个阶段仍然比较同步, 仔细收集的样品有足够的食物发展到这个 timepoint, 和每样动物的数量是可管理的 (见下文)。另外一个同步步骤对于 L3 幼虫是必要的, 因为在 L3 开发过程中发生的大量昆虫蜕皮激素脉冲对新陈代谢产生了戏剧性的影响, 并且会在不同步的种群中生成工件。</li>
	<li>用解剖针轻轻地将酵母糊从琼脂上提起。把酵母放在一个新的糖蜜琼脂帽上。 注意: 大多数幼虫会在糖蜜琼脂和酵母膏之间的界面上进食。</li>
	<li>使用小画笔收集新暴露的糖蜜琼脂表面的幼虫。将幼虫放入冰上的1.5 毫升离心管中。 注: 每个发育阶段的适当样本大小如下:50 头龄幼虫 (L1);25 mid-L2 幼虫;20早期 L3 幼虫;15 mid-L3 或晚 L3 幼虫。</li>
	<li>在收集大样本集时, 在小批量 (四到六个样本) 中清洗和冻结样品 (步骤2.3 到 2.8)。样品必须在放置幼虫的20分钟内冷冻在试管中。 注: 我们建议使用离心1.5 毫升弹性管, 因为其他管可能会干扰在步骤3的样品转移。样品必须收集在离心管。不要收集步骤3.1 中描述的珠子管中的样品。陶瓷珠保留 NaCl 洗涤液, 导致质量测量不准确, 并将污染物引入样品中。</li>
	<li>将1毫升的冰冷0.9% 氯化钠加入管中, 关闭盖子, 垂直翻转管子彻底清洗幼虫。</li>
	<li>把管子放回冰上 ~ 三十年代。在这段时间里, 幼虫会沉到管子的底部, 但酵母会保持悬浮。一旦所有幼虫形成一个松散的颗粒, 去掉氯化钠溶液使用1毫升吸管。</li>
	<li>重复步骤2.4 和步骤2.5 两次, 或直到最终清洗解决方案是明确的。 注: 如果收集的样品含有过量的酵母, 可能需要额外的洗涤步骤。</li>
	<li>将样品在 2000 x g上离心1分钟, 摄氏4摄氏度。</li>
	<li>使用200µL 吸管去除所有残留溶液。</li>
	<li>立即将样品冷冻在液氮中。 注意: 在此步骤中可以暂停该协议。样品可储存3月至-80 摄氏度。</li>
</ol>3. 将样品转移到珠管上
<ol><li>每个幼虫样品必须从1.5 毫升离心管转移到2毫升 screwcap 管, 其中含有1.4 毫米陶瓷珠。为准备这一转移步骤, 使用一个无酒精标记标签的一侧2毫升 screwcap 珠管 (盖子上的标记将被销毁在步骤 4.4)。</li>
	<li>使用能够精确测量0.01 毫克的分析天平, 将标记的珠管的质量进行包装。</li>
	<li>如果幼虫样品储存在摄氏-80 摄氏度, 则在转移之前将样品管放在液氮中。</li>
	<li>使用长钳从液氮杜瓦中除去1.5 毫升的样品管。</li>
	<li>戴腈手套, 抓住冷冻管, 反转, 并大幅打击对台式的管盖, 以赶走冰冻颗粒。立即倒入一个预 tared 2 毫升 screwcap 珠管颗粒。如果球团无法取出, 则将管子回流至液氮并重复。 注意: 幼虫丸不会从所有离心管中去除。如果使用的不是推荐的管, 确定幼虫颗粒是否可以在收集样品之前脱落, 以进行分析。</li>
	<li>快速测定幼虫颗粒和珠管的组合质量。立即将样品管放入液氮中。幼虫颗粒质量将被用来规范化的 metabolomic 数据。 注意: 在此步骤中可以暂停该协议。样品可储存3月至-80 摄氏度。</li>
</ol>4. 样品提取
<ol><li>将样品管放置在-20 摄氏度台式冷却器中。</li>
	<li>添加0.8 毫升的 prechilled (-20 °c) 90% 甲醇含有2µg/毫升 succinic-d4 酸入每管。返回样品到-20 °c 台式冷却器。 注: succinic-d4 酸作为内部标准。仅使用 HPLC 级 H2O 和甲醇。由于甲醇和其他有机溶剂是挥发性的, 难以准确测量使用空气置换吸管, 我们建议使用正位移吸管的这一步骤和所有后续步骤。</li>
	<li>通过添加0.8 毫升的 prechilled (-20 °c) 90% 甲醇, 将含有2µg/毫升 succinic-d4 酸, 放入空珠管中, 建立负控制。</li>
	<li>融汇样品30秒在6.45 米/秒使用一个珠轧机均质机, 位于4°c 温度控制室。 注意: 未能迅速完全融汇幼虫颗粒是新陈代谢分析中常见的变异来源。只使用高压, 能够在三十年代内摧毁冰冻幼虫组织。</li>
	<li>将匀质样品管返回到-20 °c 台式冷却器, 并在-20 摄氏度冷藏库中孵化至少1小时。</li>
	<li>离心管在 2万 x g或最大速度为5分钟在4°c 去除结果沉淀。</li>
	<li>将上清的600µL 转化为新的1.5 毫升离心管。不要扰乱沉淀。如果沉淀颗粒在吹打上清时脱落, 返回所有上清到管并重复步骤4.6。</li>
	<li>将样品管放在真空离心机中。在密封离心机之前, 要确保所有管子都开着。在室温下干燥样品, 直到除去所有溶剂 (这个步骤通常需要16小时完成)。 注意: 如有必要, 可以在此步骤暂停协议。干燥样品可储存在-80 摄氏度。</li>
</ol>5. 化学衍生化
<ol><li>如果干燥样品储存在-80 摄氏度, 将未开封的样品管放在真空离心机中, 干燥30分钟。此步骤必须在打开取样管之前执行。 注意: 这一步去除从冷冻库中取出的离心管外的冷凝。协议的这一部分对 H2非常敏感, 必须采取额外的预防措施, 以使所有试剂尽可能干燥。</li>
	<li>
在无水吡啶中制备40毫克/毫升 methoxylamine 盐酸盐 (混合氧化物) 溶液。只使用无水吡啶。在干燥中储存混合氧化物和吡啶, 并每天准备混合氧化物溶液。 注意: 氧化物和吡啶是有毒的。在通风罩里准备这个溶液。<ol>
<li>用热枪干1毫升的玻璃注射器。</li>
		<li>将注射器针插入无水吡啶瓶中。除去1毫升吡啶, 加入含有40毫克混合氧化物的离心管。</li>
		<li>用氩气冲洗瓶无水吡啶。把瓶子密封起来, 回到干燥。</li>
		<li>在35摄氏度的热搅拌机中, 在600转每分钟10分钟内, 将混合氧化物溶解在吡啶中。</li>
	</ol>
</li>
	<li>在无水吡啶溶液中加入40µL 40 毫克/毫升混合氧化物的干燥样品。</li>
	<li>漩涡为十年代和简要离心机 (1万 x g二十年代)。</li>
	<li>在热混合器中以 600 rpm 在30摄氏度孵育1小时。</li>
	<li>离心机在 2万 x g或最大速度为5分钟去除微粒物质。</li>
	<li>将25µL 的清液转移到一个 autosampler 瓶中, 用250µL 停用的玻璃 microvolume 插入。</li>
	<li>添加40µL n-甲基-n-trimethylsilyltrifluoracetamide (MSTFA), 其中包含1% 差旅管理公司。 注意: MSTFA 是有毒的。在通风罩中执行此步骤。 注意: 如果可用, 此步骤可以由 Gerstel autosampler 完成。</li>
	<li>用卷发工具将瓶盖放在 autosampler 瓶上并密封。</li>
	<li>在37摄氏度孵育样品1小时, 晃动 (250 rpm)。</li>
	<li>准备一个脂肪酸甲酯标准 (FAMES) 解决方案, 以前 desceribed3。在注射前, 使用机器人 autosampler 在 autosampler 瓶中添加3µL FAMES。 注: FAMEs 用于校准固定时间移位并检查仪器性能。如果没有可用的机器人 autosampler, 则可以在步骤5.8 中添加 FAMEs。</li>
</ol>6. GC-MS 检测
注: 在大多数情况下, 用户将在一个质谱核心设施的协助下执行这一步骤。此协议设计用于与 30 m, GC 列与一个 5 m 卫兵专栏。
<ol><li>随机抽样顺序。</li>
	<li>
准备 GC-MS。
	<ol>
<li>将氦载流子气体流量设置为1毫升/分。</li>
		<li>将入口温度设置为250摄氏度。</li>
		<li>程序 GC 以执行以下温度渐变:<ol>
<li>初始温度到95°c 以保持1分钟。</li>
			<li>将温度提高到110摄氏度, 速度为40摄氏度/分钟, 保持2分钟。</li>
			<li>增加到摄氏250摄氏度/分钟坡道。</li>
			<li>增加到330°c 以25°c/分钟的速度, 最后举行4分钟。</li>
		</ol>
</li>
		<li>将溶剂延迟设置为3.5 分钟。 注: 根据 GC-MS 系统可以改变时间。这一步骤的目的是防止 MSTFA 和混合氧化物破坏探测器。</li>
	</ol>
</li>
	<li>将衍生物样品的1µL 注入 GC-MS (分离率为 10:1)。 注: 如果峰值强度过低, 则注入2µL。样品的注射顺序应该是随机的。</li>
	<li>在 50–500 m/z 的质量范围内, 在全扫描模式下操作质谱仪。</li>
</ol>7. 数据分析
<ol><li>使用目标或不相关方法分析 metabolomic 数据。目标分析的重点是测量一组定义的代谢物的丰度, 如下面我们描述的乳酸测量。相比之下, 不相关分析使用一种无偏见的方法来识别在两个样本集之间明显改变的任何代谢特征。 注意: 我们的实验室主要使用免费程序 MetAlign17和 MetaboAnalyst18,19进行数据分析。由于对质量控制、规范化和数据处理步骤的充分描述超出了本手稿的范围, 因此, 我们将用户推荐给用于数据处理20、21的更详细的协议, 22。此外, 还可以在8的其他地方找到描述此分析所需步骤的流程图。</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatography/mass+spectrometry+in+metabolic+profiling+of+biological+fluids.">Gas chromatography/mass spectrometry in metabolic profiling of biological fluids.</a> Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 871 (2), 202-211 (2008).</li><li>Want, E. J., Nordstrom, A., Morita, H., Siuzdak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+exogenous+to+endogenous:+The+inevitable+imprint+of+mass+spectrometry+in+metabolomics.">From exogenous to endogenous: The inevitable imprint of mass spectrometry in metabolomics.</a> Journal of Proteome Research. 6 (2), 459-468 (2007).</li><li>Garcia, A., Barbas, C., Metz, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metabolic Profiling: Methods and Protocols Vol. 708 Methods in Molecular Biology. , 191-204 (2011).</li><li>Chan, E. C. Y., Pasikanti, K. K., Nicholson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+urinary+metabolic+profiling+procedures+using+gas+chromatography-mass+spectrometry.">Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry.</a> Nature Protocols. 6 (10), 1483-1499 (2011).</li><li>Dunn, W. B., 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=Procedures+for+large-scale+metabolic+profiling+of+serum+and+plasma+using+gas+chromatography+and+liquid+chromatography+coupled+to+mass+spectrometry.">Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.</a> Nature Protocols. 6 (7), 1060-1083 (2011).</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 Manual. , 171-178 (1989).</li><li>Biyasheva, A., Do, T. V., Lu, Y., Vaskova, M., Andres, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glue+secretion+in+the+Drosophila+salivary+gland:+a+model+for+steroid-regulated+exocytosis.">Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis.</a> Developmental Biology. 231 (1), 234-251 (2001).</li><li>Lommen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetAlign:+Interface-driven,+versatile+metabolomics+tool+for+hyphenated+full-scan+mass+spectrometry+data+preprocessing.">MetAlign: Interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing.</a> Analytical Chemistry. 81 (8), 3079-3086 (2009).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+MetaboAnalyst+3.0+for+comprehensive+metabolomics+data+analysis.">Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis.</a> Current Protocols in Bioinformatics. 55, (2016).</li><li>Xia, J., Sinelnikov, I. V., Han, B., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetaboAnalyst+3.0-making+metabolomics+more+meaningful.">MetaboAnalyst 3.0-making metabolomics more meaningful.</a> Nucleic Acids Research. 43 (W1), W251-W257 (2015).</li><li>Lommen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+(pre-)processing+of+nominal+and+accurate+mass+LC-MS+or+GC-MS+data+using+MetAlign.">Data (pre-)processing of nominal and accurate mass LC-MS or GC-MS data using MetAlign.</a> Methods in Molecular Biology. 860, 229-253 (2012).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+MetaboAnalyst+3.0+for+comprehensive+metabolomics+data+analysis.">Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis.</a> Current Protocols in Bioinformatics. 55, (2016).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+inference+of+biological+patterns,+functions+and+pathways+from+metabolomic+data+using+MetaboAnalyst.">Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst.</a> Nature Protocols. 6 (6), 743-760 (2011).</li><li>Li, H., Tennessen, 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=Methods+for+studying+the+metabolic+basis+of+Drosophila+development.">Methods for studying the metabolic basis of Drosophila development.</a> Wiley Interdisciplinary Reviews Developmental Biology. 6 (5), (2017).</li></ol>

作者没有什么可透露的。

新陈代谢提供了一个无与伦比的机会来调查代谢反应, 构成中间代谢。然而, 这种技术的敏感性使数据易受遗传背景、发育线索和各种环境应力的影响, 包括温度、湿度、人口密度和养分供应。因此, 高质量和可重复的新陈代谢分析要求在高度控制的条件下收集样品。虽然一些评论强调这一点3,8,23, 在这里我们提供了一个分步方法收集幼虫, 旨在确保重现性。
在新陈代谢分析中, 最常见的变异来源是在采集样品后, 没有淬火或停止代谢反应。在这方面, 当样品在-20 摄氏度甲醇中匀质时, 新陈代谢将停止在液氮的结冰和新陈代谢的酵素将被毁坏。假设用户特别注意在甲醇提取之前保持样品冻结, 我们的协议旨在确保此过程生成的新陈代谢数据准确地反映幼虫新陈代谢。如果用户遇到不可接受的数据变异性级别, 用户应重新检查收集和代谢物提取协议, 并给予特别注意, 以确保 (1) 新陈代谢迅速淬火 (步骤 2.9–4.2) 和 (2), 即样品在90% 甲醇 (步骤 4.4) 有效地匀质。在这方面, 许多珠磨机在指定时间内为融汇样品提供了足够的力量, 我们鼓励用户使用以前研究中使用的仪器8。
我们的协议还突出了新手用户必须注意的关键步骤, 以便重现性 derivatize 示例。这对于气相色谱-MS 是必不可少的, 因为许多代谢物具有有限的挥发性或热稳定性较差。衍生化增加了许多代谢物的挥发性和热稳定性, 从而增加了可重现性测量的化合物的数量。因此, 经历了不完全衍生的样品将展出不可重现的峰值区域、高度和形状。在这里我们强调, 失败的衍生品的一个共同的来源是暴露样品对水, 减少化学衍生效率。因此, 所有试剂, 包括 MSTFA, 氧化物, 和吡啶, 必须保持在无水条件。保持干燥条件的需要也延伸到准备储存在-80 摄氏度的样品, 在开口前应放在真空离心机中30分钟, 以确保冷凝不进入样品。我们还要强调, GC-MS 是一种敏感的技术, 因此, 较大的样本不一定会产生更好的数据。我们的协议提供实验性测试样本大小, 并将重现性测量大多数代谢产物的中央碳代谢。其中一些代谢物非常丰富, 样本量的增加将导致频谱中的信号饱和。事实上, 我们的分析中已经饱和了海藻糖的信号, 必须用100:1 的分离率来精确测量这种化合物。最后, 用户应该注意到, 因为我们使用极性提取解决方案, 我们的协议是不适合的脂肪提取。我们的协议也不考虑可能存在于塑料管和尖端的脂肪酸污染物, 这就是为什么一些脂肪酸信号出现在负控制样本中 (图 1B)。
最后, 此处详述的方法为研究果蝇代谢提供了有力的工具。但是, 此协议并不特定于果蝇, 而且可以用于在任何小无脊椎动物中进行代谢研究的少量修改。不管物种如何, 这种简单的方法允许相对定量的几乎所有氨基酸, 中间体在糖酵解和 TCA 循环, 以及其他一些小极性分子。当与果蝇社区的无与伦比的遗传工具结合使用时, 这种类型的 metabolomic 分析在可预见的将来将苍蝇置于新陈代谢研究的前沿。

果蝇是一种研究调节中间代谢的分子机制的理想系统. 不仅是大多数代谢通路保存在果蝇和人类之间, 但关键的营养传感器和生长调节剂, 如胰岛素, Tor, 和 c-myc, 也活跃在飞1,2。因此,果蝇可以用来探索人类疾病的代谢基础, 从糖尿病和肥胖到变性和癌症。在这方面,果蝇幼虫的发育提供了一个理想的框架, 其中研究代谢计划称为有氧糖酵解, 或华宝效应。正如许多肿瘤使用有氧糖酵解产生碳水化合物的生物量, 所以做果蝇幼虫激活有氧糖酵解促进发展增长3,4,5。这些幼虫与肿瘤代谢的相似性建立了果蝇作为理解如何调节有氧糖酵解的关键模型体内。
尽管苍蝇已经成为研究新陈代谢的流行模型, 但大多数的果蝇研究依赖于用于测量单个代谢产物3(如海藻糖、甘油三酯或 ATP) 的方法。由于需要一个特定的协议来衡量每个代谢产物, 以化验为基础的研究是劳动密集型的, 昂贵的, 并偏向于那些可以用商业工具来衡量的化合物。这些限制的解决方案已经出现在新陈代谢领域, 它提供了研究果蝇新陈代谢的更有效和无偏见的方法。与基于检测的研究不同, 单 metabolomic 分析可以同时测量数以百计的小分子代谢物, 并全面了解生物体的新陈代谢状况6,7。这种技术大大扩大了果蝇代谢研究的范围, 并代表了这一新兴领域8的未来。
Metabolomic 研究主要采用三技术: (一) 核磁共振 (NMR), (ii.) 液相色谱-质谱 (LC), (iii.) 气相色谱-质谱 (gc-ms)9。每种方法都具有明显的优缺点, 所有这些技术都已用于成功研究果蝇代谢。由于我们实验室进行的研究集中在小的极性代谢物上, 我们主要采用了基于 GC-MS 的方法。GC-MS 为用户提供了许多优点, 包括高重现性, 峰值分辨率, 灵敏度, 以及标准电子撞击 (EI) 光谱库的有效性, 它允许快速识别发现的代谢功能10,11。但是, GC-MS 的样品的制备有点复杂, 需要仔细注意细节。样品必须收集, 洗涤, 称量和冷冻的方式, 迅速止渴代谢反应。此外, 苍蝇胴体对标准均匀化协议有抗药性, 需要一个珠磨来确保最佳的代谢产物萃取。最后, 在检测12之前, 用 GC-MS 分析的样品必须经过化学衍生。虽然以前发布的方法描述了所有这些步骤3、13、14, 但仍然需要一个允许新手用户重现性生成高质量数据的可视化协议。在这里, 我们演示如何准备果蝇幼虫样品的 GC-MS 新陈代谢分析。该协议允许用户重现性测量许多组成中央碳代谢的小极性代谢物。

<table border="0" cellpadding="0" cellspacing="0" style="width:827px;" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Unsulfured blackstrap molasses</td>
			<td style="width:141px;">Good Food, INC</td>
			<td style="width:141px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td>Genesee Scientific</td>
			<td>66-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyridine</td>
			<td>EMD Millipore</td>
			<td>PX2012-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methoxyamine hydrocholoride (MOX)</td>
			<td>MP Biomedicals, LLC</td>
			<td>155405</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MSTFA with 1% trimethylchlorosilane</td>
			<td>Sigma</td>
			<td>69478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fleischmann&#8217;s Active dry yeast</td>
			<td>AB Mauri Food Inc</td>
			<td>2192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6oz Drosophila stock bottle</td>
			<td>Genesee Scientific</td>
			<td>32-130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soft tissue homogenizing mix (2 mL tubes)&#160;</td>
			<td>Omni International</td>
			<td>SKU:19-627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vial insert, 250 &#181;L deactivated glass with polymer feet</td>
			<td>Agilent</td>
			<td>5181-8872</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinic acid-2,2,3,3-d4</td>
			<td>Sigma</td>
			<td>293075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpeedVac</td>
			<td>Thermo&#160;</td>
			<td>SC210A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">o-Phosphoric acid</td>
			<td>Fisher Scientific</td>
			<td>A242-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propionic acid</td>
			<td>Sigma</td>
			<td>P5561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-Hydroxy benzoic acid methyl ester</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Ruptor</td>
			<td>Omni International</td>
			<td>SKU:19-040E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>5384000012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MultiTherm Shaker with a 24 X 12 mm block</td>
			<td>Benchmark Scientific</td>
			<td>H5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL centrifuge tube</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35 X 10 mm tissue culture dish</td>
			<td>Corning Incorporated</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GC column</td>
			<td>Phenomex</td>
			<td>ZB-5MSi</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of drosophila larval samples for gas chromatography-mass spectrometry (gc-ms)-based metabolomics

最近在新陈代谢领域的进展已建立了果蝇的果蝇, 作为研究动物代谢的强大遗传模型. 通过将大量的果蝇基因工具与调查大量中间代谢的能力结合起来, 新陈代谢方法可以揭示饮食、基因型、生命史事件和环境线索之间的复杂相互作用。此外, 新陈代谢研究可以发现新的酶机制, 并揭示以前未知的联系之间似乎完全不同的代谢途径。为了便于在果蝇社区中更广泛地使用此技术, 我们在这里提供了一个详细的协议, 描述如何准备用于气相色谱-质谱 (GC-MS) 的果蝇幼虫样品-基于 metabolomic 分析。我们的协议包括幼虫样本采集、代谢物提取、化学衍生和 GC-MS 分析的描述。该协议的成功完成将使用户能够测量小极性代谢物的相对丰度, 包括氨基酸、糖和糖酵解和 TCA 循环所涉及的有机酸。

乳酸脱氢酶 (dLDH) 突变体缺乏 dLDH 活性 4, 基因匹配控制被收集为 mid-L2 幼虫, 并按照上文所述的协议进行处理。与对照组相比, 突变幼虫在乳酸、丙酮酸和 L-2-hydroxyglutarate4中表现出显著变化。光谱是通过安捷伦 GC6890-5973i MS 系统获得的。使用我们的协议生成的 GC-MS 频谱示例显示在图 1中。海藻糖有许多可见的特征和显著的峰值, 通常代表幼虫样本中最大的峰值, 通常是饱和 (图 1A, B)。负控制示例的单个频谱显示在图 1C中。虽然负控制样本中仍有几个可见峰, 但与图 1A、B中所示的实验样本相比, 峰值的强度和数量也会降低。这些峰值主要是由柱流血、内部标准 (succinic-d4 酸)、FAMEs 和污染脂肪酸引起的。失败的示例准备将生成类似于图 1C中所示的频谱。
利用 MetAlign17对所有光谱进行预处理, 并将数据与内部标准和颗粒质量进行规范化。随后, 数据提交给 MetaboAnalyst18,19进行统计分析。主成分分析 (PCA) 清楚地表明, 这两组是相互分离的, 并且在任一组中都没有异常 (图 2A)。进一步的分析显示了已知受dLDH (图 2B)4损失影响的代谢产物的显著变化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57847/57847fig1.jpg"> 图 1: 代表性的 GC-MS 谱的果蝇幼虫提取物.(A-B)野生型 (mid-L2) 和dLDH突变体幼虫提取物的典型光谱。(C)由负控制 (NC) 示例生成的具有代表性的 GC-MS 频谱。这一光谱中的大部分峰值来自内部标准, FAMEs 和脂肪酸。(D F)与样品相比, 高浓度的样品表现为(D)丙酮酸含量升高, (E)乳酸和(F) 2-戊 (2-汞) 水平降低。<a href="/files/ftp_upload/57847/57847fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57847/57847fig2.jpg"> 图 2: 统计分析显示野生类型 ( dLDH ) 和挖空 (高) 幼虫之间的不同代谢剖面。(A) PCA 评分图。(B)代谢产物在dLDH突变体中表现出重大变化。所有数据点都是相对于小波控制的平均值绘制的, 它被调整为任意值100。在分析之前, 数据被规范化到 succinic-d4 酸内部标准和幼虫颗粒质量。显示为平均值的数据是一个标准偏差。p < 0.01 用于所有使用双尾t测试的代谢产物。<a href="/files/ftp_upload/57847/57847fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics at JoVE.com

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

此协议描述如何准备果蝇幼虫进行基于 GC-MS 的 metabolomic 分析。

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 ...

preparation-drosophila-larval-samples-for-gas-chromatography-mass

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

11.0K 次观看.  Indiana University. 此协议描述如何准备果蝇幼虫进行基于 GC-MS 的 metabolomic 分析。

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

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 ...

科研

发育生物学

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (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, ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

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 ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

果蝇黑色素血脑屏障完整性分析

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

解剖、免疫组织化学和幼虫和成年 果蝇 脑的安装用于视叶可视化

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...