Trioleylglycerol

Yeast strains were grown in synthetic medium (with 2% glucose) to stationary phase. Lipids were isolated from ∼100 OD of cells by the method of Folch et al. (1957) (link) with modifications. Briefly, lipids were extracted with chloroform: methanol (2:1) and washed with 1 M KCl. Lipid extracts were resolved by TLC on silica gel 60 plates (Merck, Darmstadt, Germany) in petroleum ether/diethyl ether/acetic acid (80:20:1) and visualized with methanolic MnCl₂, followed by plate charring. Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD) after digital scanning. Standards included trioleylglycerol, dioleylglycerol, and cholesterol stearate (Sigma-Aldrich).

Incorporation of [³H]oleate into TAG and SE species was quantified as previously described (Oelkers et al., 2000 (link)). Log-phase cultures cultured in YPD were pulsed with [³H]oleate (1 μCi/ml) for 30 min at 30ºC. Total lipids were extracted, resolved by TLC, and stained with iodine vapor. TAG and SE species were identified using trioleylglycerol and cholesterol stearate (Sigma-Aldrich) as standards and individually harvested, and radiolabel in each fraction was determined by liquid scintillation counting.