After appropriate treatment, cells were incubated at 37° C for the indicated time and reaction was stopped by placing the cells on ice. Floating cells were collected in chilled 13 × 100 mm glass tubes by centrifugation at 500 g for 5 min. Cells were washed once with cold PBS, and lipids were extracted by incubating cells in 0.5 ml of CH3OH for 10 minutes. Cell lysates were then added to the pelleted floating cells. Ceramide levels were analyzed using liquid chromatography, electrospray ionization-tandem mass spectrometry as described previously .