Axioscope 2microscope

Cells of the COS-1 line that endogenously express P2Y receptors coupled to Ca²⁺ mobilization and CHO,
were transfected with P2X2/P2X3 expression construct and used as cellular sensors for monitoring ambient nanomolar ATP and ADP
concentrations. The bath solution for cellular physiology experiments contained (mM) 140 NaCl, 2.5 KCl, 1 MgSO₄, 1.3
CaCl₂, 1.2 NaH₂PO₄, 10 glucose, 5 pyruvate, 10 HEPES–NaOH, pH 7.4. For calcium
imaging ATP-sensitive cells were preloaded with 4 mM Fluo-4AM+1.5 mg/ml Pluronic (both from Molecular Probes) for 30 min
at 23–25°C. Cell fluorescence was excited with a computer controlled light emitting diode (Luxion) at 480 nm and
recorded at 535 nm. Sequential fluorescence images were acquired every 0.5–2 seconds using a fluorescent Axioscope-2
microscope, an EMCCD Andor iXON camera (Andor Technology) and Workbench 6.0 software (INDEC Biosystems).
Cells were stimulated by bath application of compounds. All chemicals were from Sigma-Aldrich. Experiments were carried
out at 23–25°C.

Taste cells were assayed with the patch-clamp technique using the perforated patch (with 400 mg/l amphotericin B in the
recording pipette) or whole-cell configuration. Ion currents were recorded, filtered, and analyzed using an Axopatch 200B
amplifier, a DigiData1322 interface, and the pClamp8 software (Axon Instruments). Intracellular solution contained (mM) 100 CsCl,
40 KCl, 1 MgATP, 1 EGTA, 10 HEPES–NaOH, pH 7.4. The bath solution contained (mM) 140 NaCl, 2.5 KCl, 1 MgSO₄,
1.3 CaCl₂, 1.2 NaH₂PO₄, 10 glucose, 5 pyruvate, 10 HEPES–NaOH, pH 7.4.
For calcium imaging cells were loaded with 4 μM Fluo-4AM or FURA-2AM +1.5 mg/ml Pluronic (both from
Molecular Probes) for 30 min at 23–25°C. For Fluo-4 loaded cells fluorescence was excited with a computer
controlled light emitting diode (Luxion) at 480 nm and recorded at 535 nm. Sequential fluorescence images were acquired every
0.5–2 seconds using a fluorescent Axioscope-2 microscope, an EMCCD Andor iXON camera (Andor Technology) and Workbench 6.0
software (INDEC Biosystems). To measure Fura-2 signals, recordings were done using a VisiChrome monochromator and VisiView
software (Visitron Systems) on an AxioExaminer.D1 microscope (Zeiss) equipped with a CoolSnap HQ² camera
(Photometrics). Cells were stimulated by bath application of compounds. All chemicals were from Sigma-Aldrich. Experiments were
carried out at 23–25°C.