Pe conjugated donkey anti mouse igg

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)).