Measurement of S1P secretion by apoptotic cells. (A) Fragment spectra of S1P and the internal standard C17-S1P by using product ion scans in the positive ionization mode. Product ion scans were obtained by infusion of 100 to 1000 ng/mL of the respective analyte in methanol with a flow of 10 μL/min. (B-C) Jurkat cells were cultured in FCS-free medium, remained as controls (VCM), or were treated with 0.5 μg/mL staurosporine with (ACM-DMS) or without (ACM) the previous addition of DMS for 3 hours. After changing medium, incubation continued for another 2 hours and conditioned media were analyzed for the S1P content with liquid chromatography-tandem mass spectrometry. (B) Exemplary chromatograms of S1P representing 22.0, 42.1, and 12.2 ng/mL. (C) Quantification of S1P release in the supernatant of Jurkat cells. Data are presented as the mean ± SD from 6 independent experiments. Significant differences between samples are marked with an asterisk (P < .05).