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

Protocol
Part of the Methods in Molecular Biology book series

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 

References

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    Kobayashi N, Kobayashi N, Yamaguchi A, Nishi T (2009) Characterization of the ATP-dependent sphingosine 1-phosphate transporter in rat erythrocytes. J Biol Chem 284(32):21192–21200. doi:10.1074/jbc.M109.006163 CrossRefPubMedPubMedCentralGoogle Scholar
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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biochemistry, Faculty of Pharmaceutical SciencesSetsunan UniversityHirakataJapan
  2. 2.Department of Biomolecular Science and Regulation, Institute of Scientific and Industrial ResearchOsaka UniversityIbarakiJapan
  3. 3.Faculty of Pharmaceutical ScienceOsaka UniversitySuitaJapan

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