Basic methods for monitoring intracellular Ca2+ in cardiac myocytes using Fluo-3

Virginie Bito, Karin R. Sipido*, Niall Macquaide

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

In cardiac myocytes, the physiological increase of intracellular calcium, the [Ca(2+)]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 [Ca(2+)]i transient and, depending on the nature of the Ca(2+) signals under study, a variety of different probes are available for monitoring changes in intracellular Ca(2+). 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 Ca(2+), but the fluorescence increases dramatically on Ca(2+) binding. Although Fluo-3 is a single excitation-emission dye, it has a number of advantages for investigators, including an ideal dissociation constant (Kd) 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 [Ca(2+)]i transient with this probe during simultaneous patch-clamp and whole-cell recording of membrane voltage or of ionic currents under voltage clamp.

Original languageEnglish
Pages (from-to)392-397
Number of pages6
JournalCold Spring Harbor protocols
Volume2015
Issue number4
DOIs
Publication statusPublished - 1 Apr 2015

Keywords

  • Aniline Compounds/metabolism
  • Animals
  • Calcium/metabolism
  • Calibration
  • Coloring Agents/metabolism
  • Cytological Techniques/methods
  • Electrophysiological Phenomena
  • Fluorescence
  • Intracellular Space/metabolism
  • Myocytes, Cardiac/metabolism
  • Patch-Clamp Techniques
  • Xanthenes/metabolism

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