Fluorescent Human Chemokine (C-X-C motif) Receptor 2 (CXCR2) Internalization Assay Cell Line
A novel U2OS/tGFP-CXCR2 cell line has been developed through stable transfection to modulate the internalization of the chemokine (C-X-C motif) receptor 2 (CXCR2) complex in living cells and to assay for compounds inducing tGFP-CXCR2 distribution inside the cells.
Fluorescent CXCR2 Internalization Cell Line was obtained by transfection of an expression vector for a fusion protein of tGFP and human CXCR2, as well as puromycin into U2OS cells. Single cells with strong green fluorescence were selected by flow cytometry, and allowed to expand. These cells constitutively express the tGFP-CXCR2fusion protein.
Assay Details: Innoprot CXCR2 Internalization Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate chemokine (C-X-C motif) receptor 2 activation and the following redistribution proce Beta inside the cells. This highly reproducible assay allows monitoring CXCR2 activation and redistribution process in High Content Analysis and fluorescence microscope applications.
This internalization assay was validated with an average of Z=0.73 +/- 0.02 for High Content Screening.
Fig 1: Our expression plasmid containing the coding sequence of human chemokine (C-X-C motif) receptor 2 (Interleukin 8 receptor beta) 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 IL8R Beta /CXCR2 stimulated with IL8. Concentrations from 0 to 100 mg/l were tested for 24 h. Activation and internalization processes were detected and analyzed using BD Pathway 855 High-Content Bioimager from BD Biosciences.
Fig 3:U2OS cells, stably expressing human Interleukin 8 receptor beta tagged in the N-terminus with tGFP protein, were stimulated with increasing concentrations of IL8 during 24 h. After the treatment an accumulation of fluorescence was observed around nucleus.Nuclei were stained with DAPI and CXCR2 fluorescence redistribution was determined measuring the increase of fluorescence surrounding the nuclei using image analysis algorithms.