Synergy h4

Prior to spectral analysis, all proteins were purified using metal chelating Ni-NTA chromatography and dialyzed into PBS. Emission spectra were recorded with a Biotek Synergy H4 plate reader. Absorbance measurements were made on a Varian Cary 50 UV/Vis Spectrophotometer using a 1 cm quartz microcell cuvette. The alkaline chromophore denaturation method was used to determine ε values. mCitrine (Φ = 0.76) was used as the reference for quantum yield determination for ddOFP. pH titrations were performed by incubating purified proteins in buffers of desired pH and acquiring emission spectra with a 96-well Biotek Synergy H4 plate reader. A 1 μM solution of fluorescent protein was prepared in PBS and diluted 1:10 with a universal buffer of desired pH. This universal buffer solution was prepared by mixing equal volumes of 0.04 M H₃BO₃, 0.04 M CH₃COOH, and 0.04 M H₃PO₄. The pH was adjusted to the desired value by adding 1M NaOH to the prepared stock solution. The pK_a was determined by fitting the experimental data to the equation: $F=A+B{\left(1+{10}^{\left(p{K}_a-pH\right)n_H}\right)}^{–1}$ , where F is fluorescence, A and B are variables that define the baselines, and nH is the Hill coefficient. Mean integrated emission peak intensities were normalized and plotted as a function of pH.

HEK293 cells were plated in 96-well dishes in 100 μl of growth medium. When cells reached 70–80% confluence, they were transfected with 50 ng β₁AR-WT or β₁AR-WT + β₁AR-Mut (25 ng:25 ng) plasmids together with 50ng pGloSensor-22F (Promega) which produce rapid and reversible cAMP-dependent activation of luciferase activity. After incubation for an additional 20–24 hours, growth medium was replaced with 100μl equilibration medium (88% CO₂-independent medium, 10% FBS, 2% GloSensor cAMP Reagent stock solution (Promega)). Cells were then equilibrated for 2 hours at room temperature. Baseline luminescence was measured every min for 10 min (synergy H4, Bio Tek). 10nM isoproterenol was then added to the medium, and Luminescence was immediately measured every min for 30 min (synergy H4, Bio Tek).

Optical density of samples was quantified by measuring the absorbance at 600 nm either in a ThermoFisher Genesys 20 spectrophotometer with a 10 mm path length or in a Biotek Synergy H4 plate reader. 1 mL of culture was used for spectrophotometer measurements, and 150 µL of culture in a 96 well plate was used for plate reader measurements. Calibration curves were made for each instrument and used for uniform reporting of optical density. All optical densities reported correspond with those taken in the spectrophotometer.
For RFP quantification, fluorescence at 585 nm excitation and 610 nm emission was measured on a Biotek Synergy H4 plate reader. All reported fluorescence values are background-subtracted and normalized to optical density.

Briefly, WM266–4 and M14 cells were plated in 384-well plates in 5 μL of complete media (EMEM and RPMI-1640, respectively). 5 μL of 200 μM 2155–14 and 2155–18 were added to the cells. Plates were incubated at 37 °C, 5% CO₂ and 95% relative humidity for various lengths of time. After incubation, 5 μL of caspase 3/7, caspase 8, and caspase 9 Glo® reagent (Promega cat#: G7570) were added to each well, and incubated for 15 min at room temperature. Luminescence was recorded using a Biotek Synergy H4 multimode microplate reader. In the case of caspase 6, 5 μL of 100 μM caspase 6 substrate AFC-VEID in lysis buffer was added and incubated for 1 h at 37 °C, 5% CO₂ and 95% relative humidity and fluorescence intensity was measured at λ_excitation = 400 nm and λ_emission = 505 nm using a Biotek Synergy H4 multimode microplate reader.