Fluoromax fluorimeter

Vesicles (10–15 μl) were added to Buffer A (final volume 2 ml)) in a plastic fluorimeter cuvette equipped with a magnetic flea. The final concentration of vesicles was ~100 μM phospholipid. For β1AR proteoliposomes and paired liposomes, 1 mM isoproterenol was included in the assay buffer. Time-based NBD fluorescence was monitored using a temperature-controlled Horiba Fluoromax fluorimeter equipped with an injection port. The following settings were used: λ_ex 470 nm, λ_em 530 nm, excitation and emission slits 2 nm, cuvette temperature 25°C, stirring rate 900 rpm, data acquisition frequency 1 Hz, recording. period 1000 s. After being allowed to stabilize for 2–3 min, fluorescence was recorded for ~100 s before adding 40 μl of 1 M sodium hydrosulfite (Sigma 157953-5G) freshly prepared in 1 M Tris pH 10. All recordings were normalized to the average value recorded over the initial ~100 s and analyzed in GraphPad Prism (version 9.1.0) by fitting to a one-phase exponential decay equation with a linear component (for liposome samples; the slope of the linear component was invariably weak (<10⁻⁴ s⁻¹)) or a two-phase exponential decay equation (for proteoliposome samples), both constrained to a starting fluorescence value of 1.