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.