Fluorescent Formyl Peptide Receptor 2 (FPRL1) Internalization Assay Cell Line
A novel U2OS/tGFP-FPRL1 cell line has been developed through stable transfection to modulate the internalization of the Human Formyl peptide receptor 2 (FPRL1) complex in living cells and to assay for compounds inducing tGFP-FPRL1 redistribution inside the cells.
Fluorescent FPRL1 Internalization Assay cell line was obtained by stable transfection of an expression vector for a fusion protein of tGFP and human Formyl peptide 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-FPRL1 fusion protein.
Assay Details: Innoprot's FPRL1 Internalization Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate kFormyl peptide receptor 2 activation and the following redistribution proce Beta inside the cells. This highly reproducible a Betaay allows monitoring FPRL1 receptor activation and redistribution process in High Content Analysis and fluorescence microscope applications.
This internalization assay was validated with an average of Z=0.70 +/- 0.02 for High Content Screening.
Fig 1: Our expression plasmid containing the coding sequence of human Formyl peptide 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 FPR2/ALX stimulated with WKYMVm. Concentrations from 0 to 1 g/ml were tested for3h. Activation and internalization process were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.
Fig 3:Concentration response curve fo rWKYMVm in Formyl peptide receptor 2 cell line. Cells were treated with 12 log dilution series (n=6). The Ec50 for WKYMVm was 1.5x10-5 g/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 (1 g/ml). The internalization assay was validated with an average of Z =0.7+/-0.02 for High Content Screening.