A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

Cell Calcium. 2012 Aug;52(2):170-81. doi: 10.1016/j.ceca.2012.05.010. Epub 2012 Jun 19.


The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds / pharmacology*
  • Animals
  • Calcium / metabolism*
  • Cells, Cultured
  • Esters
  • Fluorescent Dyes / pharmacology*
  • Heart Ventricles / cytology
  • Microscopy, Confocal
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / drug effects*
  • Myocytes, Cardiac / metabolism*
  • Rats
  • Xanthenes / pharmacology*


  • Aniline Compounds
  • Esters
  • Fluo 4
  • Fluorescent Dyes
  • Xanthenes
  • Fluo-3
  • Calcium