Fluorescent Glucagon-like Peptide 2 Receptor (GLP2R) Internalization Assay Cell Line

Applications: Cell Screening
Categories: Cells

A novel U2OS/tGFP-GLP2R cell line has been developed through stable transfection to modulate the internalization of the Glucagon like peptide 2 Receptor (GLP2R) complex in living cells and to assay for compounds inducing tGFP-GLP2R redistribution inside the cells. Fluorescent GLP2R Cell Line was obtained by stable transfection of an expression vector for a fusion protein of tGFP and human GLP2R, as well as G418 into U2OS cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP-GLP2R fusion protein.

Assay Details: Innoprot GLP2R redistribution Assay kit has been designed to assay compounds or analyze stimuli for their ability to modulate Glucagon like peptide 2 Receptor activation and the following redistribution process inside the cells.

This highly reproducible assay allows monitoring GLP2R activation and redistribution process in High Content Analysis and fluorescence microscope applications. This internalization assay was validated with an average Z prime of 0.74 +/- 0.02 for High Content Screening.

Fig 1: Our expression plasmid containing the codingsequence of human Glucagon like peptide 2Receptor (GLP2R) tagged in the N-terminalwith tGFP protein. Our plasmid wastransfected in U2OS cells. Resistant clones wereobtained by limit dilution, and receptor geneexpression was tested by RT-PCR.

Fig 2: Internalization of GLP2R stimulated with GLP-2Concentrations from 0 to 1 ?M were tested for 1h. Activation andinternalization processes were detected and analyzed using BDPathway 855 High-Content Bioimager from BD Biosciences.

Fig 3: Concentration response curve for GLP-2 inGlucagon like peptide 2 receptor cell line. Cellswere treated with 10 log dilution series (n=5). The Ec50 forGLP-2 was approx 2.67x10-9M after a treatment of 1 h with theagonist. Cells were fixed and the nuclei were stained withDAPI. Percent Activity was calculated relative to positive (1?M). The internalization assay was validated with an average Z prime of 0.74+/- 0.02 for High Content Screening.