Poly d lysine coated 96 well plates

We used the Nano-Glo Live Cell Assay System (Promega. Madison, WI) for the split luciferase reconstruction assay. HEK293T cells were plated on poly-d-lysine–coated 96-well plates (Thermo Fisher Scientific). Plasmid DNAs were transfected using Lipofectamine 2000 (Invitrogen). Then, each Rtp1S was subcloned into the pBiT1.1-C (TK/LgBiT), pBiT2.1-C (TK/SmBiT), pBiT1.1-N (TK/LgBiT), and pBiT2.1-N (TK/SmBiT) vectors. Combinations of plasmids for RTP1S fusion variants were transfected into HEK293T cells. After 24 h post-transfection, the medium was replaced with 40 μl of 10 mm HEPES, and we followed the manufacturer's protocols for measuring luciferase activities. Luminescence was measured using a GloMax-Multi Detection System (Promega).

The Dual-Glo^® luciferase assay (Promega, Madison, WI, USA) was used to measure the ligand response of OR. Firefly luciferase, driven by a cAMP response element promoter (CRE-Luc; Stratagene, San Diego, CA, USA), was used to measure the OR activation levels. Renilla luciferase, driven by a constitutively active SV40 promoter (pRL-SV40), was used as an internal control for cell viability and transfection efficiency. HEK293T cells were plated on poly-D-lysine-coated 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA). Plasmid DNAs of each protein and two luciferase constructs were transfected. At 24 h post-transfection, the medium was replaced with 25 µL of odorant solution diluted in CD293 (Thermo Fisher Scientific) and incubated for 3 h at 37 °C. We followed the manufacturer’s protocols for measuring firefly luciferase (Luc) and Renilla luciferase (RL) activities.

The Dual-Glo luciferase assay system (Promega) was used for the luciferase assay (15 (link)). HEK293T cells were plated on poly-d-lysine–coated 96-well plates (Thermo Fisher Scientific). Plasmid DNAs of ORs and RTP1S were transfected using Lipofectamine 2000 (Invitrogen). In addition, two luciferase constructs were used, including a firefly luciferase gene driven by a cAMP-response element (CRE-Luc) and a Renilla luciferase gene driven by a constitutively active SV40 promoter (pRL-SV40) that was used as an internal control for cell viability and transfection efficiency. For each 96-well plate, 10 ng of CRE-Luc, 5 ng of pRL-SV40, 5 ng of OR, and 5 ng of RTP1S were transfected. Twenty four hours post-transfection, the medium was replaced with 25 μl of odorant solution diluted in CD293 (Invitrogen) and incubated for 3.5 h at 37 °C in 5% CO₂. We followed the manufacturer's protocols for measuring firefly luciferase (Luc) and Renilla luciferase (RL) activities. Luminescence was measured using a GloMax-Multidetection System (Promega). The relative response of ORs was calculated as (Luc/RL − Luc/RL_min)/(Luc/RL_max − Luc/RL_min), where Luc/RLN represents the mean value from the replicate wells of a certain sample, and Luc/RLmin represents the mean value from the minimal response in the experiment.