Measurement of Cation Movement in Primary Cultures Using Fluorescent Dyes

Ian J. Reynolds1

1 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.11
DOI:  10.1002/0471142301.ns0711s04
Online Posting Date:  May, 2001
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Abstract

Ca2+, Na+, K+, and Mg2+ have a central role in neuronal excitability. The concentration of these cations in the cytoplasm of neurons (generically termed [ion+]i) provides a marker of the excitation state of the neurons, and may also illuminate the activity of specific signaling mechanisms that involve Ca2+‐ or Mg2+‐activated enzymes. The measurement of [ion+]i in cultured neurons is achieved with the use of an ion‐sensitive fluorescent dye in combination with equipment designed to quantitatively measure fluorescence. Specificity is obtained by choosing dyes with the appropriate selectivity for the ion of interest. Measurements of steady state ion concentrations can be made, as well as measurements of the net difference between ion movement into the cytoplasm (in response to a stimulus) and the physiological buffering of that ion. The procedures in this unit for loading and recording from dyes are broadly similar for each ion when ratiometric dyes are used as described, and can readily be modified for use with single‐wavelength dyes. Support protocols are provided for calibration of individual dyes, which can be more problematic.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Measurement of [ion+]i Using Ratiometric Dyes
  • Calibration for Ratiometric Dyes
  • Support Protocol 1: Cell‐Free Calibration of Ca2+ Dyes
  • Support Protocol 2: In Situ Calibration of Ca2+Dyes
  • Support Protocol 3: In Situ Calibration of Na+ Dyes
  • Support Protocol 4: Cell‐Free Calibration of Mg2+ Dyes
  • Alternate Protocol 1: Calibration and Measurement Using Nonratiometric [Ca2+]i Dyes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of [ion+]i Using Ratiometric Dyes

  Materials
  • 1 mM dye solution (see recipe): acetoxymethyl (AM) ester of ion‐sensitive dye
  • HEPES buffer (see recipe), with and without 5 mg/ml BSA
  • Primary cultures of neurons prepared on no. 1 glass coverslips
  • Drugs for stimulating ion changes (optional): e.g., 1 mM glutamate, glycine, or N‐methyl‐D‐aspartate (NMDA) in HEPES buffer
  • 35‐mm disposable plastic culture dishes
  • Fluorescence recording system (see , Fig. )

Support Protocol 1: Cell‐Free Calibration of Ca2+ Dyes

  Materials
  • 1 mM dye solution (see recipe): pentapotassium salt of dye in deionized water
  • Ca2+ calibration solutions (see recipe and Table 7.11.2)
  • Clean coverslips (without neurons)
  • Curve‐fitting software (e.g., Prism, GraphPad Software)

Support Protocol 2: In Situ Calibration of Ca2+Dyes

  Materials
  • Intracellular buffer (see recipe)
  • 1 mM dye solution( recipe): acetoxymethylester (AM) of dye to be calibrated
  • BSA
  • 1 mM antimycin in methanol
  • 1 mM nigericin in ethanol
  • 100 mM 2‐deoxyglucose (2‐DG) in water
  • 100 mM EGTA (see recipe)
  • Primary culture of neurons prepared on no. 1 glass coverslips
  • 1 mM carbonyl cyanide p‐trifluoromethoxyphenyl hydrazone (FCCP) in methanol
  • 1 mM Ca2+ ionophore (ionomycin or 4bromo‐A23187; Molecular Probes) in dimethyl sulfoxide (DMSO)
  • 140 mM CaCl 2 in H 2O
  • 35‐mm tissue culture disk
NOTE: This approach relies on poisoning the neuron during the dye loading process to prevent Ca2+ buffering, and then using an ionophore to load the neuron with a high concentration of Ca2+.

Support Protocol 3: In Situ Calibration of Na+ Dyes

  Materials
  • HEPES buffer( recipe)
  • 1 mM SBFI acetoxymethyl ester (SBFI AM; Molecular Probes) in dimethyl sulfoxide (DMSO)
  • BSA
  • Primary culture of neurons prepared on no. 1 glass coverslips
  • Na+‐free HEPES buffer
  • 1 mM gramicidin D in DMSO
  • HEPES buffer containing 5, 10, 50, 100, and 140 mM Na+ (prepare by mixing HEPES buffer with Na+‐free HEPES buffer)

Support Protocol 4: Cell‐Free Calibration of Mg2+ Dyes

  • Ca2+‐free HEPES buffer (see recipe) containing 1 mM EGTA
  • 1 mM Ca2+ ionophore (ionomycin or 4Br‐A23187; Molecular Probes) in dimethyl sulfoxide (DMSO)
  • 100 mM CaCl 2 in H 2O
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Figures

Videos

Literature Cited

   Blinks, J.R. 1992. Intracellular [Ca2+] measurements. In The Heart and Cardiovascular System (H.A. Fozzard, ed.) pp. 1171‐1201. Raven Press, New York.
   Grynkiewicz, G., Poenie, M., and Tsien, R.Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440‐3450.
   Harootunian, A.T., Kao, J.P.Y., Eckert, B.K., and Tsien, R.Y. 1989. Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes. J. Biol. Chem. 264: 19458‐19467.
   Haugland, R.P. 1993. Intracellular ion indicators. In Fluorescent and Luminescent Probes for Biological Activity (W.T. Mason, ed.) pp. 34‐43. Academic Press, London.
   Iatridou, H., Foukaraki, E., Kuhn, M.A., Marcus, E.M., Haugland, R.P., and Katerinopoulos, H.E. 1994. The development of a new family of intracellular calcium probes. Cell Calcium. 15: 190‐198.
   Ishijima, S., Sonoda, T., and Tatibana, M. 1991. Mitogen‐induced early increase in cytosolic free Mg2+ concentration in single Swiss 3T3 fibroblasts. Am. J. Physiol. 261: C1074‐C1080.
   London, R.E. 1991. Methods for measurement of intracellular magnesium: NMR and fluorescence. Annu. Rev. Physiol. 53: 241‐258.
   Minta, A., Kao, J.P.Y., and Tsien, R.Y. 1989. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264: 18171‐18178.
   Murphy, E. 1993. Measurement of intracellular ionized magnesium. Miner. Electrolyte Metab. 19: 250‐258.
   Neher, E. 1995. The use of fura‐2 for estimating Ca buffers and Ca fluxes. Neuropharmacology 34: 1423‐1442.
   Poenie, M. 1990. Alteration of intracellular fura‐2 fluorescence by viscosity: A simple correction. Cell Calcium 11: 85‐91.
   Rizzuto, R., Simpson, A.W.M., Brini, M., and Pozzan, T. 1992. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358: 325‐327.
   Stout, A.K., Li‐Smerin, Y., Johnson, J.W., and Reynolds, I.J. 1996. Mechanisms of glutamate‐stimulated Mg2+ and subsequent Mg2+ efflux in rat forebrain neurones in culture. J. Physiol. 492: 641‐657.
   White, R.J. and Reynolds, I.J. 1995. Mitochondria and Na+/Ca2+ exchange buffer glutamate‐induced calcium loads in cultured cortical neurons. J. Neurosci. 15: 1318‐1328.
   Zhao, M., Hollingworth, S., and Baylor, S.M. 1996. Properties of tri‐ and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers. Biophys. J. 70: 896‐916.
Key Reference
   Mason, W.T. 1993. Fluorescent and Luminescent Probes for Biological Activity. Academic Press, London.
  This is an excellent book that covers all aspects of the principles and practice of fluorescence microscopy. It also discusses cation‐sensitive dyes and numerous other subjects.
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