Fluorescent Chemokine (C-C motif) Receptor 2 (CCR2) Internalization Assay Cell Line
A novel U2OS/tGFP-CCR2 cell line has been developed through stable transfection to modulate the internalization of the Human Chemokine (C-C motif) receptor 2 (CCR2) complex in living cells and to assay for compounds inducing tGFP-CCR2 redistribution inside the cells.
Fluorescent CCR2 Internalization Assay cell line was obtained by stable transfection of an expression vector for a fusion protein of tGFP and human Chemokine (C-C motif) receptor 2, 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-CCR2 fusion protein.
Assay Details: Innoprot's CCR2 Internalization Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate Chemokine (C-C motif) receptor 2 activation and the following redistribution process inside the cells. This highly reproducible assay allows monitoring CCR2 receptor activation and redistribution process in High Content Analysis and fluorescence microscope applications.
This internalization assay was validated with an average of Z=0.64 +/- 0.02 for High Content Screening.
Fig 1: Our expression plasmid containing the coding sequence of human Chemokine (C-C motif) receptor 2 tagged in the N-terminal with tGFP protein. Our plasmid was transfected in U2OS cells. Resistant clones were obtained by limit dilution, and receptor gene expression was tested by RT-PCR.
Fig 2: Internalization of CCR2 stimulated with human MCP-1.Concentrations from 0 to 10 g/ml 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 human MCP-1 in CCR2 cell line. Cells were treated with 12 log dilution series (n=6). The Ec50 for human MCP-1 was 0.73 ng/ml after a treatment of 3 h with the agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (10 g/ml). The internalization assay was validated with an average of Z =0.64+/- 0.02 for High Content Screening.