Fluorescent Cannabinoid Receptor 2 (CB2) Internalization Assay Cell Line

Applications: Cell Screening
Categories: Cells

A novel CHOK1/tGFP-CB2 cell line has been developed through stable transfection to modulate the internalization of the cannabinoid receptor 2 (CB2, CNR2) complex in living cells and to assay for compounds inducing tGFP-CB2 distribution inside the cells. Fluorescent CB2 Internalization Assay Cell Line was obtained by stable transfection of an expression vector for a fusion protein of tGFP and human CB2, 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-CB2 fusion protein.

Assay Details: Innoprot CB2 Internalizacion Assay Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate cannabinoid receptor 2 internalization process following and quantifying the fluorescence distribution inside the cells.

This highly reproducible assay allows monitoring CB2 receptor internalization process in High Content Analysis and fluorescence microscope applications.

Fig 1: Our expression plasmid containing the codingsequence of human Cannabinoid receptor 2tagged in the N-terminal with tGFP protein. Our plasmid was transfected in CHO-K1 cells,using calcium phosphate method. Resistantclones were obtained by limit dilution, andreceptor gene expression was tested by RTPCR.

Fig 2: Internalization of CB2-tGFP clone3 andclone5 stimulated with CP-55940. Cells were treatedwith 100nM CP-55940 for 16h. Activation andinternalization processes were detected and analyzedusing BD Pathway 855 High-Content Bioimager fromBD Biosciences.

Fig 3: CP55940 concentrations response in theCB2R-tGFP internalization assay. Cells were treatedwith 8 log dilution series (n=8). The Ec50 for the CP55940was approx 47nM after a treatment of 16h with agonist. Cellswere fixed and the nucleus were stained with DAPI. Percent Activity was calculated relative to positive (1uM). Theinternalization assay was validated with an average Z prime of 0.91+/- 0.01 for High Content Screening.