Fig. 4.

Effect of BSA and KCl on the fluorescence intensity of the aqueous phase containing NBD-S1P. Erythrocytes were incubated in buffer A containing 0.1% (A–D) or 1% BSA (A, B) with 5 μM NBD-Sph at 37°C. After incubation for 60 min (60 min) or without incubation (0.5 min), the cell suspensions were centrifuged briefly. The lipids in the supernatant (extracellular buffer) were extracted under alkaline conditions according to the procedures described in the Materials and Methods (A, B) and with/without KCl (C, D). The fluorescence intensity of the aqueous phase was measured using a fluorescence microplate reader in a 96-well plate (A, C). For NBD-S1P quantitation, 50 μl of the aqueous phase was dried and analyzed by TLC (B, D). The fluorescence intensities of the aqueous phase and the extracellular NBD-S1P prepared from the samples containing 0.1% BSA (60 min) (A, B) or extracted by the addition of 1.5 M KCl (60 min) (C, D) were set to 100%. ND, not detected. The experiments were repeated three times, and the error bars indicate the SD.