Mass spectrometry to measure sphingolipid levels

For lipid extraction, cells were washed with ice-cold PBS, then directly lysed with 2 ml of cell extraction mixture (2:3 70% isopropanol/ethyl acetate) followed by gentle scraping of the cell from the culture plate. Small intestine tissues were directly homogenized in a cell extraction mixture using FastPrep-24 from MP Biomedicals (Santa Ana, CA, USA). Lysate and homogenate were then transferred to 15-ml Falcon tubes. Lipid extracts were analyzed by the Lipidomics Core Facility at Stony Brook University Medical Center. Data were normalized by total inorganic phosphate present in the sample (30) after a Bligh and Dyer extraction (31). Sphingolipid levels were expressed as picomoles per nanomole of inorganic phosphate.