A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes.
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.
Center for Biomedical Engineering and Technology & Department of Physiology, University of Maryland, Baltimore, 21201, USA.
Brian M
BM
Hagen
Liron
L
Boyman
Joseph P Y
JP
Kao
W Jonathan
WJ
Lederer
D000814:Aniline Compounds / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004952:Esters; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006352:Heart Ventricles / Q000166:cytology; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats; D014966:Xanthenes / Q000494:pharmacology*
D000814:Aniline Compounds; D004952:Esters; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium