Fluorescent Bradykinin Receptor 2 (BDKR2) Internalization Assay Cell Line

Applications: Cell Screening
Categories: Cells

A novel CHOK1/tGFP-BDKR2 cell line has been developed through stable transfection to modulate the internalization of the bradykinin receptor 2 (BDKR2) complex in living cells and to assay for compounds inducing tGFP-BDKR2 distribution inside the cells. BDKR2 Internalization Cell Line was obtained by transfection of an expression vector for a fusion protein of tGFP and human BDKR2, as well as G418 into CHOK1 cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP-BDKR2 fusion protein.

Assay Details: Innoprot BDKR2 Internalizacion Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate bradykinin receptor 2 internalization process following and quantifying the fluorescence distribution inside the cells.

This highly reproducible assay allows monitoring BDKR2 receptor internalization process in High Content Analysis and fluorescence microscope applications.

Fig 1: Our expression plasmid containing the coding sequence of human Bradykinin receptor 2 tagged in the N-terminal with tGFP protein. Our plasmid was transfected in CHO-K1 cells, using calcium phosphate method. Resistant clones were obtained by limit dilution, and receptor gene expression was tested by RT-PCR.

Fig 2: Internalization of BDK2-tGFP stimulated with Bradykinin. Cells were treated with 1uM Bradykinin for 6h. Activation and internalization processes were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.

Fig 3: Bradykinin concentrations response in the BDK2R-tGFP internalization assay. Cells were treated with 8 log dilution series (n=8). The Ec50 for the Bradykinin was approx 52nM after a treatment of 6h with agonist. Cells were fixed and the nucleus were stained with DAPI. Percent Activity was calculated relative to positive (1uM). The internalization assay was validated with an average Z prime of 0.63+/- 0.01 for High Content Screening.