Effects of sphingosine on PS exposure, osmotic fragility, and intracellular calcium in WT RBCs. (A) Flow cytometry for annexin V on WT RBCs treated with different sphingosine concentrations (1 µM or 10 µM) or vehicle (ethanol, control) or for several time points as indicated. The FL2 channel was empty. (B) Osmotic fragility in WT RBCs (n = 3) pretreated with different sphingosine (Sph) concentrations or vehicle (ethanol) for 6 hours. (C) FACS plots of WT RBCs preloaded with Fluo-4 and exposed to Ringer with sphingosine (1 µM or 10 µM) or vehicle (ethanol, control) for 30 minutes. The FL2 channel was empty. (D) Osmotic fragility in WT RBCs (n = 3) pretreated with vanadate (1 mM) or vehicle (H2O) for 1 hour at 37°C using solutions with decreasing concentrations of NaCl. **P < .01 and ***P < .001 for untreated vs treated RBCs (2-way ANOVA followed by Bonferroni post hoc analysis for panels B,D).
