Intracellular Ca2+ measurements in live cells by rapid line scan confocal microscopy: simplified calibration methodology.
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 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 interpretaton and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be used to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.
Plank
David M
DM
SDSU Heart Institute, San Diego State University, Department of Biology, San Diego, CA 92182, USA.
Sussman
Mark A
MA
D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D014966:Xanthenes / Q000737:chemistry
D000814:Aniline Compounds; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium