Biochemical and high throughput microscopic assessment of fat mass in Caenorhabditis elegans.
Biochemical and high throughput microscopic assessment of fat mass in Caenorhabditis elegans.
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DOI：
10.3791/50180
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发表时间：
2013-03-30
期刊：
影响因子：
--
通讯作者：
Soukas AA
中科院分区：
文献类型：
--
作者：
Pino EC;Webster CM;Carr CE;Soukas AA
线虫C.秀丽线虫已经成为研究调节脂肪代谢的保守遗传途径的重要模型，因为它与人类肥胖及其相关的病理学有关。几个以前的方法开发的可视化C。已经证明，秀丽隐杆线虫富含磷脂酰胆碱的脂肪储存是错误的，突出了脂滴以外的细胞区室。其他方法需要专门的设备，耗时，或产生不一致的结果。我们介绍了一种快速、重复性好、固定液为基础的尼罗红染色方法，用于准确、快速地检测C.优美的在40%异丙醇中的短暂固定步骤使动物完全渗透尼罗红，然后用于染色动物。这种亲脂性染料的光谱特性使其仅在富含脂质的环境中才能在黄绿色光谱中强烈和选择性地发出荧光，但在极性更强的环境中则不然。因此，在异丙醇中仅进行简短的尼罗红染色步骤后，就可以在配备有简单GFP成像能力的荧光显微镜上观察脂滴。该方案的速度、经济性和可重复性使其非常适合高通量筛选。我们还展示了一个配对的方法，使用气相色谱质谱生化测定甘油三酯和磷脂。该更严格的方案应用作尼罗红显微镜脂质测定结果的确认。我们期望这些技术将成为C语言领域的新标准。elegans代谢研究。
The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.
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