M1000 multimode plate reader

To measure the affinities of association between β1AR and EGFR constructs, U2OS cells seeded in 96-well plates were initially infected with adenoviruses encoding Flag-EGFR-mYFP versus Flag-EGFR-CT230-mYFP and Flag-β1AR-mCFP at increasing MOIs. 24 h post-infection, the cells were rinsed and media replaced with imaging buffer (HBSS buffer (Cellgro), 0.2% bovine serum albumin, 20 mM HEPES) for 3 hr, after which cyan fluorescent protein (CFPex/CFPem), yellow fluorescent protein (YFPex/em) and FRET (CFPex/YFPem) readings were attained using a M1000 multimode plate reader (Tecan). Quantification of FRET (corrected FRET (cFRET) = FRET − (CFP*CFP bleedthrough) − (YFP*YFP bleedthrough) were expressed as a percentage of total CFP emission (%FRET = cFRET/[cFRET + CFP]). Plate reader fluorescence bleedthrough values were: 40% (CFP) and 2% (YFP). Nonlinear regression one-site binding analysis was used to calculate maximal % FRET (FRET_max) and affinity (10/K_FRET) values for mCFP/mYFP association (Graphpad Prism 8.4.3). In subsequent stimulation experiments, cells were infected with Ad-Flag-EGFR-mYFP and Ad-Flag-β1AR-mCFP at MOIs indicated to achieve a baseline FRET value of ~10% for evaluation of pharmacologic disruption of β1AR-EGFR association with gefitinib (1 μM), JMD peptides (10 μM) or vehicle (1% DMSO). Following treatment, fluorescence values were attained every 5 min for up to 1 hr.

U2OS cells were seeded in either 96 well plates or 35 mm glass-bottom dishes (MatTek Corporation) coated with 10 μg/ml fibronectin. For βarr2 recruitment assays the cells were infected with adenoviruses encoding βarr2-mYFP (MOI of 10) and either Flag-β1AR-mCFP (MOI of 10) or Flag-β2AR-mCFP (MOI of 20). The cells underwent pretreatment with vehicle, paroxetine or fluoxetine (30 μM each) for 45 min, following which they were rinsed and media replaced with imaging buffer (HBSS buffer (Cellgro), 0.2% bovine serum albumin, 20 mM HEPES) and stimulated with buffer or ISO (100 nM). FRET detection was performed using either a M1000 multimode plate reader (Tecan) for 96 well plates or a Leica DMI4000B inverted microscope with a Leica DFC365 FX 1.4-megapixel monochrome digital camera for 35 mm plates. Cyan fluorescent protein (CFPex/CFPem), yellow fluorescent protein (YFPex/em) and FRET (CFPex/YFPem) readings were measured every 60 seconds (plate reader) or 3.6 seconds (microscope). Quantification of FRET (corrected FRET (cFRET) = FRET − (CFP*CFP bleedthrough) − (YFP*YFP bleedthrough) were expressed as a percentage of total CFP emission (%FRET = cFRET/[cFRET + CFP]). Microscope bleedthrough values were: 36% (CFP) and 13% (YFP); plate reader bleedthrough values were: 40% (CFP) and 2% (YFP).