Cell culture and transfection
Human embryonic kidney
(HEK293T) cells were maintained in a humidified incubator at 37°C with
5% CO2 and grown to 90-95% confluence in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U
ml-1 penicillin and 100 µg ml-1streptomycin. Cells were transfected using Lipofectamine 2000 according
to the manufacturer’s recommended protocol. All transfections were
performed under serum-free conditions in Opti-MEM. Transfections were
allowed to proceed for 4-5 h before the media was changed back to DMEM
with 10 % FBS. Experiments were run 24 h after transfection. For cAMP
assays, cells were plated in 6-mm dishes. DNA was kept constant at 6 µg
and 6 µl of Lipofectamine2000 per plate was used. Empty vector
(pcDNA3.1+) was used to adjust the total amount of DNA. The lack of cAMP
stimulation in control vector (pcDNA3.1+) transfected cells and the
robust change in cAMP levels in D1/NOP transfected cells after treatment
with specific ligands were used to establish the success of transfection
(Feng et al. , 2017). This is
possible because HEK293T cells do not natively express D1 and NOP
receptors.