Fluorescent Dopamine Receptor 1 (DRD1) Internalization Assay Cell Line
A novel SH-SY5Y/tGFP-DRD1 cell line has been developed through stable transfection to modulate the internalization of the Dopaminergic receptor D1 (DRD1) complex in living cells and to assay for compounds inducing tGFP-DRD1 distribution inside the cells.
Fluorescent DRD1 cell line was obtained by transfection of an expression vector for a fusion protein of tGFP and human DRD1, 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 express the tGFP-DRD1 fusion protein.
Assay Details: Innoprot DRD1 Internalizacion Assay Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate dopaminergic receptor D1 internalization process following and quantifying the fluorescence distribution inside the cells.
This highly reproducible assay allows monitoring DRD1 receptor internalization process in High Content Analysis and fluorescence microscope applications.
Fig 1: Our expression plasmid containing the coding sequence of human Dopaminergic receptor D1 tagged in the N-terminal with tGFP protein.Our plasmid was transfected in SH-SY5Y cells.Resistant clones were obtained by limitdilution, and receptor gene expression was tested by RT-PCR.
Fig 2: Internalization of DRD1 stimulated with Dopamine. Concentrations from 0 to 100 M were tested for 1h. 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 D1 receptor cell line. Cells were treated with 7 log dilution series (n=8). The Ec50 for the Dopamine was 5.25x10-6M 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=0.67+/- 0.02 for High Content Screening.