Bacteria total lipid extraction

Total lipids were extracted from S. aureus using the method of Bligh and Dyer [73]. Briefly, overnight cultures were diluted 1:100 into 50 mL RPMI, with or without GTO or BCFA precursor supplementation and grown for 8 hours at 37°C with shaking. Bacteria were washed three times with 0.9% NaCl, followed by centrifugation at 5,000 × g for 15 minutes at 4°C and storage at −80°C. For lipid extraction, the pellets were mixed with 10 mL methanol (Fisher)-chloroform (Alfa Aesar) solution (2:1, vol/vol) and incubated at room temperature on a shaker for 2 hours. The lipid-containing layer was isolated after centrifugation at 5,000 × g for 15 minutes at 4°C. The extraction was repeated twice using 10 mL methanol-chloroform-water solution (2:1:0.8, vol/vol/vol). 7 mL of chloroform and 7 mL of water were added in sequential order to the lipid solution in a separatory funnel and separation was allowed to occur overnight. The lipid-containing layer was collected the next day and dried under nitrogen gas.