Quantification of Cellular Ceramides

To assess the levels of cellular ceramides, human HaCaT KC were pretreated with IL-4 (50 ng/mL) for 20 h, followed by incubation with exogenous PS (25 μM) with or without 10 μM of CB1 inhibitor (AM-251, Tocris Bioscience, Ellisville, MO, USA) for 4 h. Extraction of ceramides was performed as we have reported previously [23 (link)]. The extracted lipids were dried using a vacuum system (Vision, Seoul, Korea), re-dissolved in methanol, and analyzed by LC-ESI-MS/MS (API 3200 QTRAP mass, AB/SCIEX, Framingham, MA, USA) in the multiple reaction monitoring (MRM) mode. The ceramide MS/MS transitions (m/z) were 510→264 for C14-ceramide, 538→264 for C16-ceramide, 552→264 for C17-ceramide, 566→264 for C18-ceramide, 594→264 for C20-ceramide, 648→264 for C24:1-ceramide, 650→264 for C24-ceramide, 676→264 for C26:1-ceramide, and 678→264 for C26-ceramide, respectively. Data were acquired using the Analyst 1.5.1 software (Applied Biosystems, Foster City, CA, USA). Ceramide levels are expressed in pmol per mg protein.