Basic Methods for Monitoring Intracellular Ca²⁺ in Cardiac Myocytes Using Fluo-3

Abstract

In cardiac myocytes, the physiological increase of intracellular calcium, the [Ca²⁺]_i transient, elicited during excitation–contraction coupling typically reaches a peak amplitude of up to 1 µm, from a resting value of ∼100 nm, within 50–100 msec, depending on the species. Various conditions will affect the amplitude and rise time of the [Ca²⁺]_i transient and, depending on the nature of the Ca²⁺ signals under study, a variety of different probes are available for monitoring changes in intracellular Ca²⁺. In this protocol, we focus on Fluo-3, which exists in the cytosol in its salt form K₅Fluo-3. This form is practically nonfluorescent in the absence of Ca²⁺, but the fluorescence increases dramatically on Ca²⁺ binding. Although Fluo-3 is a single excitation–emission dye, it has a number of advantages for investigators, including an ideal dissociation constant (K_d) value and high quantum yield, meaning that it can be used at low concentrations that introduce minimal buffering. Here, we describe the basic setup and methodology for recording the global cytosolic [Ca²⁺]_i transient with this probe during simultaneous patch-clamp and whole-cell recording of membrane voltage or of ionic currents under voltage clamp.