Anti rhodopsin 4d2 antibody

HEK293T cells were seeded in 35-mm dishes and then transfected with 1200 ng of an OR expression plasmid and 300 ng of an RTP expression plasmid per dish. A total of 30 ng of a GFP expression plasmid was also transfected per dish as a control for transfection efficiency. At 24 h post-transfection, the cells were dissociated in Cellstripper (Corning, Corning, NY) and washed with staining buffer (PBS containing 2% (v/v) fetal bovine serum and 15 mm NaN₃). Then, the cells were transferred to a tube for incubation with the anti-rhodopsin 4D2 antibody (Merck Millipore, Billerica, MA) for 45 min on ice and then with phycoerythrin (PE)-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min on ice. Each antibody was diluted in staining buffer. To stain dead cells, 7-amino-actinomycin D (Merck Millipore) was added before analysis. The cells were analyzed using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA) and a BD LSRFortessa cell analyzer (BD Biosciences) with gating allowing for 10,000 GFP-positive, single, spherical, and viable cells, and the measured PE intensities were analyzed and visualized using FlowJo (15 (link), 36 (link)).

HEK293T cells were grown to confluence, resuspended, and seeded onto 35 mm plates at 25% confluency. The cells were cultured overnight and then transfected with the genes in pCI vector. GFP expression vector was also transfected per dish as a control for transfection efficiency. At 24 h post-transfection, the cells were dissociated in Cellstripper^® (CORNING, Corning, NY, USA) and transferred to a tube for incubation with the anti-rhodopsin 4D2 antibody (Merck) and APC-conjugated donkey anti-mouse IgG (BioLegend, Sandiego, CA, USA). To stain dead cells, 7-Amino-actinomycin D (Merck) was added. The cells were analyzed using CytoFLEX (Beckman Coulter, Brea, CA, USA) with gating allowing for GFP positive, single, spherical, viable cells; the measured PE intensities were analyzed and visualized using Flowjo.