Lipid analysis pmol.xlsx

2x10⁷CTRL and human neutral sphingomyelinase 2 deficient Jurkat cells were starved in RPMI/0.5ûS for 2 hrs and left unstimulated or stimulated for 10 min with the anti-CD3 (clone UCHT-1) crosslinked with the goat anti-mouse IgG (both 5 mikrog/ml). Plasma membranes were isolated by Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Inc., England) according to manufacturers' protocol. Up to 4 isolations were pooled for one plasma membrane preparation used for lipid analysis. Three preparations were analyzed for each type of cells or stimulations. Mass spectrometry-based lipid analysis was performed by Lipotype GmbH (Dresden, Germany) as described (Sampaio et al., 2011).