Cell culturing, transfection and generation of stable cell lines
COS-7 cells were cultured at 10% CO2, 95% air humidity and 37°C in Dulbecco’s Modified Eagle Medium (DMEM) 1885 supplemented with 10% fetal bovine serum (FBS), 1% penicillin (180 U/mL)/streptomycin (45 µg/mL). The cells were transfected using the calcium phosphate precipitation method as previously described (Jensen et al. 2008). Briefly, the cells were seeded in T175/T75/T25 flasks one day before transfection with 40/20/10 µg receptor DNA. Transiently transfected COS-7 cells were used in cAMP accumulation and for whole cell homologous and heterologous binding.
HEK-293 cells stably expressing hGLP-2R or pcDNA3.1(+) were generated by transfection as described above. The cells were cultured at 10% CO2, 95% air humidity and 37°C in Dulbecco’s Modified Eagle Medium (DMEM), containing 1% GlutaMAX, and supplemented with 10% fetal bovine serum (FBS) 1% penicillin (180 U/mL)/streptomycin (45 µg/mL) and 0.4 mg/mL G418 for selection. Stably transfected HEK-293 cells were utilized for membrane preparation used in kinetic binding experiments.