Bioluminescence Resonance Energy Transfer Assays

The saturation and
competition binding
assays were performed according to the methodology of Stoddart.^{18 (link)} Briefly, the assays were performed on stably
transfected NLucP2Y₂-1321N1 cells that had been seeded
24 h prior to the experiment in white Thermo Scientific Matrix 96-well
microplates. The medium in each well was removed and replaced with
HBSS containing apyrase (1 U/mL) and the required concentration of
the fluorescent ligand with or without the competing ligand. Upon
the addition of the fluorescent ligand, the cells were incubated for
1 h at 37 °C without CO₂. The NLuc substrate, furimazine
(Promega), was then added to a final concentration of 10 μM,
and the plate was incubated for a further 5 min at 37 °C without
CO₂. The luminescence and resulting BRET were measured
using a PHERAstar FS plate reader (BMG Labtech) at room temperature.
For the assays involving 97, sequential measurements
of the filtered light emissions were made at 460 nm (80 nm bandpass)
and >610 nm (long-pass), and the raw BRET ratios were calculated
by
dividing the >610 nm emissions by the 460 nm emissions. For the
assays
involving 98, the measurements were made at 475 nm (30
nm bandpass) and 535 nm (30 nm bandpass), and the raw BRET ratios
were calculated by dividing the 535 nm emissions by the 475 nm emissions.