Quantification of Ceramides and Sphingomyelins

Following lipid extraction with methanol:chloroform (2:1, v/v) as described [16 (link)], ceramides and sphingomyelins were quantified by LC–MS/MS using a 6490 triple–quadrupole mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive electrospray ionization mode (ESI +) [36 (link)]. Quantification was performed with MassHunter Software (Agilent Technologies). Sphingolipid amounts were normalized to protein content (in vivo experiments) or cell numbers (in vitro experiments. As such, concentrations per mg protein (in vivo experiments) or per 1 million cells (in vitro experiments) were calculated.