Lipid Profiling of Cells Treated with Ascorbic Acid

scr-MDA and sh-MDA cells were grown in 100 mm plates in routine medium or in routine medium supplemented with 50 μM AA as the sodium salt (Sigma) for 72 h. Cells were then washed 3 times with 0.1% BSA in ice-cold PBS, and scraped in ice-cold methanol and H₂O. Total lipids were extracted [17 (link)] and separated by TLC on silica-gel G60 plates (Merck) with hexane-ethylether-acetic acid (80:20:1; v/v/v) as the mobile phase for neutral lipid separation. All samples were chromatographed in parallel with pure lipid standards. To analyze the FA composition, total PL and TAG fractions were scraped from the plate and eluted with hexane: chloroform: methanol (3:2:1, v/v/v). FA methyl esters were obtained by reaction with BF₃ in methanol and analyzed by gas liquid chromatography in a Hewlett-Packard HP 6890 chromatograph equipped with an Omega Wax capillary column [2 (link)].