Cells were grown at 30°C to mid–log phase in 250 ml YPDA medium and collected. Total lipids were extracted by the Bligh and Dyer method (Bligh and Dyer, 1959 (link)). Phospholipid amounts were determined by phosphorus assay (Rouser et al., 1970 (link)). For the phospholipid analysis, samples containing 200 nmol phosphates were subjected to TLC plates (Merck, Darmstadt, Germany), and phospholipids were detected as described previously (Mioka et al., 2018 (link)). To detect free and esterified ergosterol, lipid extracts containing 20 nmol phosphates were subjected to high-performance TLC (Merck) separation with hexane/diethyl ether/formic acid (40:10:2, vol:vol:vol). Ergosterols were stained with a mixture of ferric chloride/sulfuric acid/acetic acid by heating (Lowry, 1968 (link)), and the spots were scanned by an image analyzer. The ergosterol content was determined by TLC-densitometric analysis using ImageJ.
Protocol detail
Quantification of Yeast Lipid Profiles
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Acetic acid
Assay
Cells
Densitometric
Diethyl ether
Ergosterol
Ferric chloride
Formic acid
Hexane
Lipid
Phosphates
Phospholipids
Phosphorus
Spots
Sulfuric acid
Corresponding Organization :
Other organizations : Hokkaido University, University of British Columbia
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Variable analysis
independent variables
- Growth temperature (30°C)
- Total lipid content
- Phospholipid amount
- Ergosterol content (free and esterified)
- Growth medium (YPDA)
- Lipid extraction method (Bligh and Dyer)
- Phospholipid quantification method (phosphorus assay)
- Phospholipid separation method (TLC)
- Ergosterol separation method (high-performance TLC)
- Ergosterol detection method (ferric chloride/sulfuric acid/acetic acid staining)
- Positive control: Not specified
- Negative control: Not specified
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