Metafectene

HEK293 cells were maintained in Minimum Essential Media (MEM, Biochrom GmbH, Berlin, Germany) supplemented with 5% fetal calf serum (FCS) and non-essential amino acids (Biochrom AG, Berlin, Germany), in humidified air at 37°C and 5% CO2. Cells were seeded in poly-L-lysine coated (Biochrom GmbH, Berlin, Germany) 96-well assay plates, at a density of 1.5 × 10⁵ cells/ml. After 24 h, cells were transiently transfected using METAFECTENE (0.45 μl/well), in supplement-free Advanced MEM (Life Technologies, Carlsbad, CA, USA). For the HiBiT assay, cells were transfected with the pcDNA3 empty vector (mock), TAAR1-WT, or co-transfected with TAAR1-WT and variants to resemble the heterozygous state, as well as GLPR as a positive control. For the GloSensor^™ cAMP assay, cells were co-transfected with TAAR1-WT and variants and the GloSensor plasmid F22 (Promega, Mannheim, Germany). As a negative control, TAAR1 was exchanged with empty vector (mock). In order to mimic the heterozygous state of the variants, TAAR1-WT and mutants were transfected in equimolar plasmid amounts.

Cells were grown at 37°C in humidified air containing 5% CO2. For sandwich ELISA, COS7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Biochrom GmbH, Berlin, Germany), supplemented with 10% fetal calf serum (FCS). 1 × 10⁶ cells were seeded in 6 cm dishes and transfected with Metafectene according to the manufactures protocol (Biontex, Munich, Germany).
HEK293 were cultured in MEM (Biochrom GmbH, Berlin, Germany) with 5% FCS and non-essential amino acids (Biochrom AG, Berlin, Germany). For FRET experiments HEK293, cells (1 × 10⁵ cells/ Ø 35 mm dish) were seeded in poly-L-lysine coated FluoroDishes (World Precision Instruments, Sarasota, FL, USA). Twenty-four hours later, the cells were transfected with 1 μg DNA per well and 4.5 μl GeneJuice (EMP Millipore Corp., Billerica, MA, USA). For cAMP and nuclear factor of activated T-cells luciferase (NFAT-luc) measurements, HEK293 cells were seeded in poly-L-lysine coated (Biochrom GmbH, Berlin, Germany) 96-well plates (1.5 × 10⁴ cells/well). Transient transfection of HEK293 cells was performed 24 h after seeding in supplement-free Advanced MEM (Life Technologies, Carlsbad, CA, USA), using Metafectene with 0.45 μg DNA per well and 0.45 μL Metafectene per well.