Pc 17 0 17 0

HepG2 and C2C12 cells were grown in 75 cm 2 flasks until ~70-80% confluent before being treated overnight with CON or DHA media. The following day cells (~80-90% confluency) were trypsinised and counted. Prostate cells (LNCaP, DU145, PC3 and PNT1) were grown to ~80-90% confluency before harvest by trypsination followed by cell counting. 1-2 million cells of each line were centrifuged (300 x g, 5 min) and resuspended in PBS containing 10% FCS and 2 mM EDTA. This process was then repeated to remove all traces of growth media. Cells were placed on ice and sorted within an hour by FACS (BD FACSAria™ III, BD Biosciences, Sydney, NSW Australia) directly into a 96 well plate preloaded with methanol spiked with 0.01% BHT and internal standards (PC 17:0/17:0 and dihydrosphingomyelin, DHSM, 12:0; Avanti Polar Lipids, Alabaster, AL, USA). Internal standards were added at rate of 1000 fmol per well for fifty cells and 1.38 fmol per single cell. Plates were sealed and stored at -80ºC until analysis. Prior to analysis methanol:chloroform containing 5 mM ammonium acetate was added to each well to achieve a final ratio of 2:1 v/v (final volume 40 μl). Empty wells containing solvent and internal standard only were included for background subtraction.