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Part of the series Methods in Molecular Biology pp 1-10

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A Rapid Fluorescence Assay for Measuring Sphingosine-1-Phosphate Transporter Activity in Erythrocytes

  • Naoki KobayashiAffiliated withDepartment of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University Email author 
  • , Tsuyoshi NishiAffiliated withDepartment of Biomolecular Science and Regulation, Institute of Scientific and Industrial Research, Osaka UniversityFaculty of Pharmaceutical Science, Osaka University Email author 

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Abstract

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.

Keywords

Sphingosine-1-phosphate Transporter Erythrocyte NBD Fluorescence 96-Well plate reader