A Rapid Fluorescence Assay for Measuring Sphingosine-1-Phosphate Transporter Activity in Erythrocytes
Sphingosine-1-phosphate (S1P) is an intercellular signaling molecule that is present in the plasma and plays an important role in recruiting lymphocytes from the thymus and secondary lymphoid organs. Erythrocytes are the most abundant cells in the blood and substantially contribute to the S1P supply in the plasma by releasing intracellularly synthesized S1P via an S1P transporter. Thus, the S1P transporter in erythrocytes is a potential target for immuno-suppressing drugs.
In this chapter, we describe a rapid method for measuring the activity of the erythrocyte S1P transporter by using the fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. This method does not require chromatography performed with high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry, or thin-layer chromatography methods. Furthermore, S1P transporter activity can be detected by measuring the increase in fluorescence intensity in the extracellular buffer without performing lipid extraction.
KeywordsSphingosine-1-phosphate Transporter Erythrocyte NBD Fluorescence 96-Well plate reader
This work was supported by JSPS KAKENHI (grant number 25870695 to N. K.) and by the Cooperative Research Program of the “Network Joint Research Center for Materials and Devices” and AMED-CREST (to N. K. and T. N.).