L glutamine sodium pyruvate

Human embryonic kidney 293 (HEK293, American Type Culture Collection CRL-1573) cells were cultured in DMEM prepared with 45g/L fetal bovine serum (FBS), 4.5g/L of penicillin, L-glutamine sodium pyruvate, and non-essential amino acid mix (Gibco) in the fully humidified incubator with constant temperature of 37°C and 5% carbon dioxide. Cell cultures were transiently transfected with wild-type SLC6A14 and YFP cDNAs (Genecopoeia, TakaraBio) using jet-PRIME transfection reagent followed by the protocol supplied by POLYPLUS-Transfection. Cells were incubated for 20–30h after transfection and ready for electrophysiology technique.

Mercury Orange (1-(4-chloromercuriophenylazo)-2-naphthol), Annexin V-FITC, DCFDA (2′,7′-dichlorofuorescein diacetate), JC-1 (1H-Benzimidazolium, 5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-, iodide), propidium iodide (PI), glyphosate (PESTANAL^®, analytical standard), imidacloprid (PESTANAL^®, analytical standard), and imazalil (PESTANAL^®, analytical standard) were obtained from Sigma-Aldrich (Merck, Germany). Versene, trypsin-EDTA, Dulbecco’s Modified Eagle Medium (DMEM), streptomycin, penicillin, L-glutamine sodium pyruvate, foetal bovine serum (FBS), and DHPE-FITC (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine) were obtained from Gibco (Alfagene, Invitrogen, Portugal). Alamar Blue^® was purchased from Invitrogen, Life-Technologies (Porto, Portugal). Caco-2 and HepG2 cell lines were purchased from Cell Lines Service (CLS; Eppelheim, Germany).