Fluorescent Acetylcholine Muscarinic Receptor 1 (M1) Internalization Assay Cell Line

Applications: Cell Screening
Categories: Cells

A novel CHOK1/tGFP-M1 cell line has been developed through stable transfection to modulate the internalization of the M1 receptor complex in living cells and to assay for compounds inducing tGFP-M1 distribution inside the cells. Fluorescent M1 cell line was obtained by transfection of an expression vector for a fusion protein of tGFP and human M1, 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-M1 fusion protein.

Assay Details: Innoprot M1 Internalizacion Assay Cell Line has been designed to assay compounds or analyze stimuli for their ability to modulate cholinergic receptor, muscarinic 1 internalization process following and quantifying the fluorescence distribution inside the cells.

This highly reproducible assay allows monitoring CHRM1 receptor internalization process in High Content Analysis and fluorescence microscope applications.

Fig 1: Our expression plasmid containing the codingsequence of human Cholinergic muscarinic 1receptor tagged in the N-terminal with tGFPprotein. Our plasmid was transfected in CHOK1cells, using calcium phosphate method.Resistant clones were obtained by limitdilution, and receptor gene expression wastested by RT-PCR

Fig 2: Internalization of M1-tGFP clone2stimulated with Oxotremorine. Cells were treatedwith 1mM Oxotremorine for 24h. Internalization processeswere detected and analyzed using BD Pathway 855High-Content Bioimager from BD Biosciences.

Fig 3: Oxotremorine concentrations response in theM1-tGFP internalization assay. Cells were treatedwith 8 log dilution series (n=4). The Ec50 for theoxotremorine was approx. 1uM after a treatment of 16h withagonist. Cells were fixed and the nucleus were stained withDAPI. Percent Activity was calculated relative to positive(1mM). The internalization assay was validated with an average Z prime of 0.6+/- 0.10 for High Content Screening.