FluorescentHuman Kappa Opioid Receptor (KOR) Internalization Assa Cell Line

Applications: Cell Screening
Categories: Cells

A novel U2OS/tGFP-KOR cell line has been developed through stable transfection to modulate the internalization of the Human Kappa Opioid Receptor (KOR) complex in living cells and to assay for compounds inducing tGFP-KOR redistribution inside the cells.

Fluorescent KOR Internalization Assay cell line was obtained by stable transfection of an expression vector for a fusion protein of tGFP and human kappa-opioid receptor, 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-KOR fusion protein.

Assay Details: Innoprot KOR Internalization Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate kappa-opioid receptor activation and the following redistribution process inside the cells. This highly reproducible assay allows monitoring KOR receptor activation and redistribution process in High Content Analysis and fluorescence microscope applications. This internalization assay was validated with an average Z prime of 0.84 +/- 0.02 for High Content Screening.

Fig 1:

Our expression plasmid containing the codingsequence of human Kappa opioid receptortagged in the N-terminal with tGFP protein.Our plasmid was transfected in U2OS cells.Resistant clones were obtained by limitdilution, and receptor gene expression wastested by RT-PCR.

Fig 2: Internalization of KOR stimulated with U-50488. Concentrations from 0 to 10 uM were tested for 2h.Activation and internalization processes were detected andanalyzed using BD Pathway 855 High-Content Bioimagerfrom BD Biosciences.

Fig 3:Concentration response curve for U-50488 in Kappa opioid receptor cell line. Cells were treatedwith 12 log dilution series (n=5). The Ec50 for the U-50488was approx 3.42x10-10M after a treatment of 2 h with the agonist.Cells were fixed and the nuclei were stained with DAPI. Percent Activity was calculated relative to positive (100?M). Theinternalization assay was validated with an average Z prime of 0.84+/- 0.02 for High Content Screening.