Mabn15

Hana3A or HEK293T cells were seeded in 6-well plates at a density of 3 × 10⁵ cells per well with 2 ml DMEM medium with 10% FBS and incubated for 24 h at 37 °C with 5% CO₂. Cells were then transfected with 0.4 μg pCI-mRTPs, 0.3 μg pEGFP-N1, and 2 μg wild-type receptor plasmid (mTAAR9 or sTAAR365) or mutant receptor plasmid, and incubated at 37 °C with 5% CO₂ for 24 h. The null pCI plasmid, pCI-mRTPs, and pEGFP-N1 were cotransfected as a negative control. Subsequently, transfected cells were dissociated with CellstripperTM (Corning) and transferred into 5 ml tubes for antibody incubation. 100 μl mouse monoclonal anti-rhodopsin antibody (MABN15, Millipore; 1:100 diluted in staining buffer: 5% BSA, 1% NaN₃ in PBS) was added to each tube and incubated at 4 °C for 45 min. Cells were washed twice by adding 2 ml staining buffer and centrifuged at 200 × g for 3 min at 4 °C. In the next step, 100 μl phycoerythrin-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch; 1:100 diluted in staining buffer: 5% BSA, 1% NaN₃ in PBS) was added and incubated at 4 °C for 30 min. Cells were washed twice by adding 2 ml staining buffer and then centrifuged at 200 × g for 3 min at 4 °C. Cell pellets were resuspended in 500 μl staining buffer for flow cytometry analysis (BD LSRFortessaTM X-20, Becton, Dickinson and Company).