Imaging B2 Receptor-Mediated Endocytosis

B₂ receptor-mediated transport of immune complexes constructed at the cell surface was previously reported (Bawolak et al., 2011 (link)). Intact HEK 293a cells stably expressing the N-terminally myc-tagged B₂ receptor construction (human sequence, Charest-Morin et al., 2015 (link)) were treated with the monoclonal anti-myc antibody (clone 4A6) conjugated with AlexaFluor-488 (Millipore, dilution 1:500) added to the culture medium. Cells were further incubated at 37°C. Without rinsing, a selected antagonist or their DMSO vehicle was added 5 min later, and BK (100 nM) or its saline vehicle 15 min later. After an additional 30 min incubation period at 37°C, cells were rinsed 3 times with Hank's balanced salt solution (Multicell Wisent, St. Bruno, Canada) and photographed in transmission and epifluorescence using an Olympus BX51 microscope coupled to a CoolSnap HQ digital camera (filters for AlexaFluor 488: excitation 460–500 nm, emission 510–560 nm).