Lv200 bioluminescence microscope

We performed bioluminescent imaging experiments to determine the localization of the Nluc-β₂AR and Rluc8-β₂AR fusion proteins. We performed all imaging experiments using the Olympus LV200 bioluminescence microscope equipped with a Hamamatsu ImagEM EMCCD camera and a 100x/1.4 UPLanSApo. We transfected HEK293 cells with expression constructs for Nluc-β₂AR and Rluc8-β₂AR, plated in 35 mm optically clear dishes (Ibidi) at a density of 200,000 cells per dish in 2 ml growth medium (DMEM supplemented with 10% FCS) and incubated for 24 h in a tissue culture incubator. We then replaced the growth medium with 1 ml OptiMEM followed by incubation for 180 min at 37°C. Immediately prior to image acquisition, we replaced the media with OptiMem including the luciferase substrates furimazine (Nluc) or coelenterazine h (Rluc8) at a final concentration of 10 μM. We identified suitable fields of view based on imaging of total luminescent signal of the donor fusion. We acquired images using an EM gain of 200 and exposure times of 1 sec (Nluc-β₂AR) and 20 sec (Rluc8-β₂AR). We acquired all images using Olympus cellSens software and performed image processing with ImageJ software.