FRET Imaging of Sensor Transduction

FRET imaging experiments were performed 24–48 h after transduction with adenovirus carrying each sensor, as described before53 (link). Cells were maintained at room temperature in a modified Ringer solution (125 mM NaCl, 20 mM Hepes, 1 mM Na₃PO₄, 5 mM KCl, 1 mM MgSO₄, 5.5 mM glucose, CaCl₂ 1 mM, pH 7.4). ARVM were imaged 18 h after infection and kept at ∼35 °C in Tyrode solution containing 1.4 mM Ca²⁺. An inverted microscope (Olympus IX71) with a PlanApoN, 60 × , NA 1.42 oil immersion objective, 0.17/FN 26.5 (Olympus, UK), was used. The microscope was equipped with a CoolSNAP HQ2 (link) monochrome camera (Photometrics) and a DV2 optical beam-splitter (MAG Biosystems, Photometrics). Images were acquired and processed using MetaFluor 7.1, (Meta Imaging Series, Molecular Devices). FRET changes were measured as changes in the background-subtracted 480 nm/545 nm fluorescence emission intensity on excitation at 430 nm and expressed as R/R₀, where R is the ratio at time t and R₀ is the average ratio of the first 8 frames. FRET imaging experiments with ARVM isolated from MI rats and age-matched controls were performed using an ORCA-ER CCD camera (Hamamatsu Photonics, Welwyn Garden City, UK) attached to an inverted microscope (Nikon TE2000) equipped with a 30 Watt dia-illuminator. The system has an EX436/20 excitation filter combined with DM455 dichroic mirror.