2.4.1. Cell culture and membrane preparation
Human embryonic kidney (HEK293) cells were used for the uptake and binding experiments. The generation and maintenance of stable, monoclonal cell lines expressing the human isoforms of DAT, SERT and the organic cation transporter 2 or 3 (OCT-2, OCT-3) was carried out as described previously (Mayer et al., 2016b, 2016a). HEK293 were maintained in DMEM supplemented with heat-inactivated 10% FBS, 100 U/ml penicillin and 100 µg/ 100 ml streptomycin, and cultured to a subconfluential state in a humidified atmosphere (5% CO2, 37ºC). Geneticin (G418; 50 µg/ml) was added to maintain the selection process.
For the uptake inhibition assays, HEK293 cells expressing different monoamine transporters were seeded at a density of 0.36 million cells per well onto poly-D-lysine (PDL) coated 96-well plates, 24h prior to the experiment.
For membrane preparations, stably transfected HEK293 cells (hDAT and hSERT) were harvested from 15-cm dishes 80-90% confluent. Briefly, cells were washed twice with ice-cold phosphate buffered saline (PBS), mechanically detached from the dish with a plastic scraper in the same ice-cold PBS and pelleted by centrifugation (400 x g for 10 minutes at 4ºC). The resulting pellet was resuspended in hypotonic HME buffer (20 mM HEPES NaOH, 2 mM MgCl2, 1 mM EDTA; pH 7.4), followed by two freeze-thaw cycles in liquid nitrogen and homogenization through sonication at 4ºC. Thereafter, membranes were collected by centrifugation (40.000 x g for 30 min at 4ºC) and resuspended in an appropriate volume of HME buffer. The membrane preparations were kept at -80ºC until use. Protein concentration was determined using the Bio-Rad Protein Reagent (Bio Rad Laboratories, Hercules, CA).