Fluorescent Somatostatin Receptor Type 3 (SSTR3) Internalization Assay Cell Line
A novel BetaTR3-tGFP/U2OS cell line has been developed through stable transfection to modulate the internalization of the Somatostatin Receptor Type 3 ( BetaTR3) complex in living cells and to assay for compounds inducing tGFP- BetaTR3 distribution inside the cells.
Fluorescent BetaTR3 Internalization Cell Line was obtained by transfection of an expression vector for a fusion protein of tGFP and human BetaTR3, as well as puromycin into U2OS cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP- BetaTR3 fusion protein.
Assay Details: Innoprot BetaTR3 Internalization Cell Line has been designed to a Betaay compounds or analyze stimuli for their ability to modulate somatostatin receptor type 3 activation and the following redistribution proce Beta inside the cells. This highly reproducible assay allows monitoring BetaTR3 activation and redistribution process in High Content Analysis and fluorescence microscope applications.
This internalization assay was validated with an average of Z=0.66 +/- 0.02 for High Content Screening.
Fig 1: Our expression plasmid containing the coding sequence of human Somatostatin receptor type 3 tagged in the N-terminal with tGFP protein. Our plasmid was transfected in U2OS cells. Resistant clones were obtained by limit dilution, and receptor gene expression was tested by RT-PCR.
Fig 2: Internalization of BetaTR3 stimulated with human Somatostatin 28.Concentrations from 0 to 10 M were tested for 3h. Activation and internalization proce Betaes were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.
Fig 3:Concentration response curve for human Somatostatin 28 in BetaTR3 cell line. Cells were treated with 8 log dilution series (n=5). The Ec50 for human Somatostatin 28 was 9.25x10-8M after a treatment of 3 h with the agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (10M). The internalization assay was validated with an average of Z =0.66+/- 0.02 for High Content Screening.