果蝇脂肪组织耗氧率测定   

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Original research article

A brief version of this protocol appeared in:
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摘要:脂肪组织作为能量调节的重要组织,其氧耗速率可以间接反映代谢状态。本实验利用药物处理结合呼吸追踪指示脂肪组织的线粒体代谢状态 (Ding等,2018)。我们反复试验发现,果蝇幼虫脂肪组织对解偶联剂FCCP几乎没有响应,因此将检测程序简化为一步处理,只检测“基础呼吸水平”和“非线粒体呼吸水平”。

关键词: 耗氧率, 果蝇, 脂肪组织, 线粒体, Seahorse

材料与试剂

  1. 果蝇幼虫
  2. Schneider's Medium (Gibco, catalog number: 10902-088)
  3. 丙酮酸钠
  4. NaOH
  5. Antimycin A (Sigma)
  6. Rotenone (Sigma)
  7. Assay Medium (AM) (见溶液配方)
  8. Antimycin A/Rotenone混合溶液 (见溶液配方)

仪器设备

  1. Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience) 
  2. 胰岛板 (Seahorse Bioscience)
  3. 探针板 (Seahorse Bioscience)
  4. 水化板 (Seahorse Bioscience)
  5. CO2培养箱

实验步骤

  1. 实验前将探针板扣于水化板 (如图1,每孔含500 μl校准液),置于预处理工作站37 °C无CO2培养箱中水化孵育过夜。


    图1. 探针板水化图示. 上部所示为24孔探针板,下部所示为探针板扣于水化板后的完整结构。

  2. 实验当天,在Assay Medium (AM) (溶液配方1)中解剖幼虫 (L3) 脂肪组织,将解剖出来的组织置于胰岛板样孔 (盛有500 μl AM),用迷你网筛将组织固定在样孔底部。建议每孔盛放不少于3只幼虫的组织。由于成虫脂肪组织解剖难度太大,从解剖收集样品到上机步骤间耗时过长,这样可能会影响组织活度,因此不建议用成虫脂肪组织进行该实验。
  3. 用AM配制Antimycin A/Rotenone混合溶液 (溶液配方2)。以每56 μl/孔将混合溶液加入加药孔中,保证最终每孔给药浓度为12 μM Antimycin A和3 μM Rotenone。
  4. 将探针板置于盛有样品的胰岛板之上,进Seahorse XF24 Extracellular Flux Analyzer进行呼吸监测,全程在室温进行,整体上机监测时长不宜超过120分钟。
  5. 呼吸监测完毕后将胰岛板取出,每个样孔里的组织独立回收进行Bradford蛋白含量测定。最终的相对耗氧率用呼吸指数除以该样品所含蛋白量表示。其中,在加药前所得曲线为“基础呼吸水平”,加药后所得稳定曲线值为“非线粒体呼吸水平”。

溶液配方

  1. Assay Medium
    Schneider's Medium加入2 mM丙酮酸钠配制,用NaOH调pH至6.5~6.8
  2. Antimycin A/Rotenone混合溶液
    949 μl AM
    48 μl 2.5 mM Antimycin A
    3 μl 10 mM Rotenone

参考文献

  1. Ding, L., Yang, X., Tian, H., Liang, J., Zhang, F., Wang, G., Wang, Y., Ding, M., Shui, G. and Huang, X. (2018). Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism. EMBO J 37(17).
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:丁隆, 黄勋. (2019). 果蝇脂肪组织耗氧率测定. Bio-101: e1010299. DOI: 10.21769/BioProtoc.1010299.
How to cite: Ding, L. and Huang, X. (2019). Determination of Oxygen Consumption Rate of Adipose Tissue in Drosophila. Bio-101: e1010299. DOI: 10.21769/BioProtoc.1010299.
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