Bioluminescence imaging

HEK293 cells stably expressing LgBiT-LDLR were plated at a density of 40,000 cells/well in 400 μl growth medium in a Labtek chambered coverslip (Nunc). Following 24 h of incubation, the growth medium was removed by aspiration and replaced with 100 μl of Opti-MEM containing 15 nM PCSK9-SmBiT and either 30 nM anti-PCSK9 antibody (BPS Biosciences) or 75 nM nonspecific control antibody (trastuzumab), followed by 60 min incubation at 37°C in a tissue culture incubator. Immediately before imaging, 25 μl of 5× Nano-Glo Luciferase Assay reagent was added to each sample. Imaging was performed by using the Olympus LV200 bioluminescence imager, which is equipped with a Hamamatsu ImagEM CCD camera, a 40×/0.9 NA objective, and a temperature-controlled stage. Images were acquired with the acquisition feature of the Olympus CellSense software package. For image acquisition, exposure times/EM (electron-multiplying) gains were set to 50 ms/0 and 15 s/1000 for the brightfield and luminescence channels, respectively. Postacquisition processing was performed with the FIJI-ImageJ package. Each bioluminescence image was generated using an average intensity Z projection of an image stack containing 15 subsequently taken images.