Fluorescent Neurokinin 1 (NK1R) Internalization Assay Cell Line
A novel SHSY5Y/ NK1R-tGFP cell line has been developed through stable transfection to modulate the internalization of the Neurokinin 1 receptor (NK1R) complex in living cells and to assay for compounds inducing NK1R-tGFP redistribution inside the cells.
Fluorescent NK1R Cell Line was obtained by transfection of an expression vector for a fusion protein of tGFP and human NK3R, as well as G418 into SHSY5Y cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP- NK1R fusion protein.
Assay Details: Innoprot NK1R Internalization Assay Cell LIne has been designed to assay compounds or analyze stimuli for their ability to modulate NK1R receptor activation and the following redistribution process inside the cells. This highly reproducible assay allows monitoring NK1R receptor activation and internalization process using High Content Analysis and fluorescence microscope applications.
This internalization assay has been validated with an average of Z=0.69+/- 0.01 for High Content Screening.
Fig 1: Our expression plasmid containing the coding sequence of Human Neurokinin 1 Receptor (NK1R) 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 TACR1 stimulated with Substance P.Concentrations from 0 to 10 M were tested for 3h. Activation and internalization processes were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.
Fig 3:Concentration response curve for Substance P in Neurokinin 1 receptor cell line. Cells were treated with 12 log dilution series (n=8). The Ec50 for the Substance P was 1,8.10-8M after a treatment of 3 h with agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (10 M). The internalization assay was validated with an average of Z =0.69 +/- 0.02 for High Content Screening.