Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
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Pino-2013-Biochemical and high.pdf
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491.41 KB
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Author(s)
Pino, Elizabeth C.
Webster, Christopher M.
Carr, Christopher E.
Soukas, Alexander A.
Date Issued
March 2013
Journal
Journal of Visualized Experiments
Publisher
MyJoVE Corporation
Citation
Pino, Elizabeth C., Christopher M. Webster, Christopher E. Carr, and Alexander A. Soukas. “Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans.” JoVE no. 73 (2013).
Version
Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Earth, Atmospheric, and Planetary Sciences
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.3791/50180
