Fluorescent Neurokinin 3 Receptor (NK3R) Internalization Assay Cell Line

Applications: Cell Screening
Categories: Cells

A novel SHSY5Y/ NK3R-tGFP cell line has been developed through stable transfection to modulate the internalization of the Neurokinin 3 receptor (NK3R) complex in living cells and to assay for compounds inducing NK3R-tGFP redistribution inside the cells. Fluorescent NK3R 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- NK3R fusion protein.

Assay Details: Innoprot NK3R Internalization Assay Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate NK3R receptor activation and the following redistribution process inside the cells. This highly reproducible assay allows monitoring NK3R receptor activation and internalization process using High Content Analysis and fluorescence microscope applications. This internalization assay has been validated with an average Z prime of 0.70+/- 0.01 for High Content Screening.

Fig 1:

Our expression plasmid containing the codingsequence of human Neurokinin3 receptortagged in the C-terminal with tGFP protein.Our plasmid was transfected in SHSY5Y cells,using calcium phosphate method. Resistantclones were obtained by limit dilution, andreceptor gene expression was tested by RTPCR.

Fig 2: Internalization of NK3R-tGFP stimulatedwith Substance P (SP). Cells were treated with 10uM SPfor 6h. Activation and internalization processes weredetected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.

Fig 3:NK3R-tGFP internalization in response to SPconcentrations. Cells were treated with 6 log dilutionseries (n=8). The Ec50 for the SP was 2.6 ?M after atreatment of 6h with agonist (Substance P). Cells werefixed and nuclei were stained with DAPI. Percent Activity wascalculated relative to positive (10uM). The internalizationassay was validated with an average Z prime of 0.7 +/- 0.1 forHigh Content Screening.