Fluorescent Calcitonin Receptor (CALCR) Internalization Assay Cell Line
A novel CHOK1/tGFP-CALCR cell line has been developed through stable transfection to modulate the internalization of the Calcitonin Receptor (CALCR) complex in living cells and to assay for compounds inducing tGFP-CALCR distribution inside the cells. Fluorescent CALCR Internalization Cell Line was obtained by transfection of an expression vector for a fusion protein of tGFP and human CALCR, as well as puromycin into CHOK1 cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP-CALCR fusion protein.
Assay Details: Innoprot CALCR Internalization Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate calcitonin receptor activation and the following redistribution process inside the cells. This highly reproducible assay allows monitoring CALCR activation and redistribution process in High Content Analysis and fluorescence microscope applications. ThIs internalization assay was validated with an average Z prime =0.60 +/- 0.02 for High Content Screening.
Fig 1: Our expression plasmid containing the codingsequence of human Calcitonin receptor taggedin the N-terminal with tGFP protein. Ourplasmid was transfected in CHO-K1 cells.Resistant clones were obtained by limit dilution, and receptor gene expression was tested by RT-PCR.
Fig 2: Internalization of CALCR stimulated with Calcitonin. Cells were treated with 100 nM Calcitonin for 3h. Activation and internalization processes were detected and analyzed using BD Pathway 855 High-ContentBioimager from BD Biosciences.
Fig 3:Concentration response curve for Calcitoninin Calcitonin receptor cell line. Cells were treated with 9 log dilution series (n=8). The Ec50 for the Calcitonin was approx 1.53 nM after a treatment of 3 h with agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (100 nM). The internalization assay was validated with an average of Z´=0.60+/- 0.02 for High Content Screening.