Fatty acid composition of isolated lipid droplets and young adult
nematodeswas determined by gas chromatography/mass spectrometry
(GC/MS) as previously described [30,31]. Separation of the TAG and
phospholipid fractions used a two-solvent TLC protocol. Lipids were extracted
by adding 5 ml of ice-cold chloroform:methanol (1:1) and incubating
overnight at 20 °C with occasional shaking. A solution of 0.2 M
H3PO4 and 1 M KCl was added to samples, which resulted in phase
separation of the organic and aqueous phase. The organic phase was
removed and dried under argon, then resuspended in chloroform.
Samples were loaded in triplicate, and TLC plates were developed two
thirds of the way up the plate in the first solvent system:
chloroform:methanol:water:acetic acid (65:43:3:2.5), dried, and then
the second solvent system hexane:diethylether:acetic acid (80:20:2)
was developed to the top of the plate. Lipids were visualized under UV
light after spraying the plate with 0.005% primuline, and spots corresponding
to TAG and the major phospholipids were scraped, spiked
with a known standard (15:0), and transesterified for GC/MS analysis
to determine the fatty acid composition as well as to determine the relative
levels of TAG, phosphatidylethanolamine (PE), and phosphatidylcholine
(PC) fractions. At least three biological replicates were used for
TLC analysis. Significance was determined by t-test calculation using
GraphPad Prism 5 software.