Fluorescent Dopamine Receptor 3 (DRD3) Internalization Assay Cell Line
A novel SH-SY5Y/tGFP-DRD3 cell line has been developed through stable transfection to modulate the internalization of the Dopaminergic receptor D3 (DRD3) complex in living cells and to assay for compounds inducing tGFP-DRD3 distribution inside the cells.
Fluorescent DRD3 Internalization Cell Line was obtained by transfection of an expression vector for a fusion protein of tGFP and human DRD3, as well as G418 into SH-SY5Y cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively expre Beta the tGFP-DRD3 fusion protein.
Assay Details: Innoprot DRD3 Internalization Assay Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate dopaminergic receptor D3 internalization process following and quantifying the fluorescence distribution inside the cells.
This highly reproducible assay allows monitoring DRD3 receptor internalization process in High Content Analysis and fluorescence microscope applications.
Fig 1: Our expression plasmid containing the coding sequence of human Dopaminergic receptor D3 tagged in the N-terminal with tGFP protein.Our plasmid was transfected in SH-SY5Y cells.Resistant clones were obtained by limit dilution, and receptor gene expression was tested by RT-PCR.
Fig 2: Internalization of DRD3 stimulated with Dopamine. Concentrations from 0 to 100 M were tested for1h. Activation and internalization processes were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.
Fig 3:Concentration response curve for Dopamine in Dopamine D3 receptor cell line. Cells were treated with 7 log dilution series (n=8). The Ec50 for the Dopamine was 3.9x10-7 M after a treatment of 1 h with the agonist.Cells were fixed and the nuclei were stained with DAPI. %Activity was calculated relative to positive (100 uM). The internalization assay was validated with an average of Z=0.79+/- 0.02 for High Content Screening.