Quantifying Intracellular Calcium Dynamics

A cohort of myocytes was loaded with fura-2/AM (0.5 μM) for 10 min and fluorescence intensity were recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix). Myocytes were placed onto an Olympus IX-70 inverted microscope and imaged through a Fluor x 40 oil objective. Cells were exposed to light emitted by a 75W lamp and passed through either a 360 or a 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480-520 nm and qualitative change in fura-2 fluorescence intensity (FFI) was inferred from FFI ratio at the two wavelengths (360/380). Fluorescence decay time was calculated as an indicator of intracellular Ca²⁺ clearing 11 (link).