Fluorescence-based Ligand Uptake Assay

A subclone of HEK 293 cells, called HEK 293a, originally obtained from Sigma-Aldrich was used in these experiments, either as a non-transfected cell type or as one stably expressing myc-tagged human B₂Rs [8 (link)]. These cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 1 % l-glutamine, and 1 % penicillin–streptomycin stock solutions (100×). Cells were detached using the protease-free Cell Dissociation Buffer (Invitrogen), incubated in D-MEM without serum at 37 °C for 30 min under agitation in the presence of a Cy7-conjugated ligands and other drugs, rapidly centrifuged (30 s, 11,000g) and resuspended in phosphate buffered saline. Then, the fluorescence of the cell suspensions was assessed using the BD SORP LSR II cell analyzer for the uptake of the as a function of stimulation and transgene expression. The cytofluorometry results were analyzed using the BD FACS DIVA software.