Fluorescent Sphingosine-1-Phosphate Receptor 1 (S1P1R) Internalization Assay Cell Line

Applications: Cell Screening
Categories: Cells

A novel HepG2/tGFP-S1P1 cell line has been developed through stable transfection to modulate the internalization of the bHuman Sphingosine-1-phosphate receptor 1 (S1P1R) complex in living cells and to assay for compounds inducing tGFP-S1P1R distribution inside the cells. Fluorescent S1P1R cell line was obtained by transfection of an expression vector for a fusion protein of tGFP and human S1P1 Receptor, as well as G418 into HepG2 cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP-S1P1R fusion protein.

Assay Details: Innoprot S1P1R redistribution Assay Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate S1P1 receptor activation and the following redistribution process inside the cells. This highly reproducible assay allows monitoring S1P1 receptor activation and redistribution process in High Content Analysis and fluorescence microscope applications. ThIs internalization assay was validated with an average Z prime of 0.71+/- 0.02 for High Content Screening.

Fig 1: Our expression plasmid containing the codingsequence of human S1P1R tagged in the Cterminalwith tGFP protein. Our plasmid wastransfected in HepG2 cells. Resistant cloneswere obtained by limit dilution, and receptorgene expression was tested by RT-PCR.

Fig 2: Redistribution of S1P1R stimulated withisoproterenol. Cells were treated with 10 uM S1P1 for h.Activation and redistribution processes were detected andanalyzed using BD Pathway 855 High-ContentBioimager from BD Biosciences.

Fig 3: S1P1 concentrations response in the S1P1Rredistribution assay. Cells were treated with 6 logdilution series (n=8). The Ec50 for the S1P was approx 0.69 ?Mafter a treatment of 1h with agonist. Cells were fixed andthe nucleus were stained with DAPI. Percent Activity wascalculated relative to positive (10 uM). The internalizationassay was validated with an average Z prime of 0.71+/- 0.02 forHigh Content Screening.