Altered intracellular Ca2+ dynamics are characteristically
observed in cardiomyocytes from failing hearts. Studies of
Ca2+ handling in myocytes predominantly use Fluo-3 AM, a
visible light excitable Ca2+ chelating fluorescent dye in
conjunction with rapid line-scanning confocal microscopy. However,
Fluo-3 AM does not allow for traditional ratiometric determination of
intracellular Ca2+ concentration and has required the use of
mathematic correction factors with values obtained from separate
procedures to convert Fluo-3 AM fluorescence to appropriate
Ca2+ concentrations. This study describes methodology to
directly measure intracellular Ca2+ levels using
inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with
Ca2+ concentration standards. Titration of Ca2+
concentration exhibits a linear relationship to increasing Fluo-3 AM
fluorescence intensity. Images obtained from individual myocyte
confocal scans were recorded, average pixel intensity values were
calculated, and a plot is generated relating the average pixel
intensity to known Ca2+ concentrations. These standard plots
can be used to convert transient Ca2+ fluorescence obtained
with experimental cells to Ca2+ concentrations by linear
regression analysis. Standards are determined on the same microscope
used for acquisition of unknown Ca2+ concentrations,
simplifying data interpretation and assuring accuracy of conversion
values. This procedure eliminates additional equipment, ratiometric
imaging, and mathematic correction factors and should be useful to
investigators requiring a straightforward method for measuring
Ca2+ concentrations in live cells using
Ca2+-chelating dyes exhibiting variable fluorescence
intensity.