Measurements of Intracellular Free Calcium Concentration in Biological Systems

Karlene K. Gunter1, Thomas E. Gunter1

1 University of Rochester, Rochester, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.5
DOI:  10.1002/0471140856.tx0205s01
Online Posting Date:  May, 2001
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Abstract

Intracellular calcium plays a role in signaling and as a second messenger in many types of cells and its concentration is closely regulated in cells. Two techniques for measuring intracellular free calcium are using fluorescent ratiometric and nonratiometric probes are described in this unit: fluorescent spectroscopy to measure calcium concentrations in a suspension of cells and fluorescent digital imaging microscopy (FDIM) to measure calcium concentrations in plated cells.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Measurement of Intracellular [Ca2+] Using Fluorescence Spectroscopy
  • Alternate Protocol 1: Measurement of FMin and FMax with Nonratiometric Probes
  • Support Protocol 1: Determination of Kd for Cells in Suspension
  • Basic Protocol 2: Measurements of [Ca2+] in Plated Cells Using Fluorescence Digital Imaging Microscopy (FDIM)
  • Support Protocol 2: Determination of Kd for Plated Cells Using Ratiometric Probes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Intracellular [Ca2+] Using Fluorescence Spectroscopy

  Materials
  • 5 µM fluorescent probe free acid (FA) in low‐Ca medium (see recipe)
  • 5 µM FA in high‐Ca medium (see recipe)
  • 2 to 5 mM AM form of fluorescent Ca2+ probe (see recipe)
  • Standard medium (see recipe), ice cold
  • 2 × 106 to 1 × 107 cell/ml suspension of sample cells in recipestandard medium
  • 1× calibration medium (see recipe)
  • 2× high‐Ca medium (see recipe)
  • Detergent: e.g., N‐octyl β‐D‐glucopyranoside
  • 2× low‐Ca medium (see recipe)
  • 10 µM FA solution (see recipe)
  • Factor that will induce changes in [Ca2+]
  • 0.1 mM thapsigargin (see recipe)
  • 2 mM oligomycin (see recipe)
  • 1 mM carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) in ethanol (store indefinitely at −20°C; use caution as compound is hazardous)
  • 1 mM ionophore (see recipe)
  • Experimental factors
  • Fluorescence spectrometer that can excite and detect in necessary wavelength ranges and has ability to constantly stir samples
  • Low‐speed centrifuge for washing cells
  • Absorption cuvettes
  • Fluorescence cuvettes
  • 37°C shaker bath
  • 4°C agitator or shaker bath
  • Cuvette stir bars, preferably star head or X‐shaped

Alternate Protocol 1: Measurement of FMin and FMax with Nonratiometric Probes

  • EGTA solution (see recipe for Ca2+ calibration standards)
  • CaEGTA solution (see recipe for Ca2+ calibration standards)
  • Ca solution (see recipe for Ca2+ calibration standards)
  • 1 M CaCl 2
  • Cells incubated with probe (see protocol 1, step )

Support Protocol 1: Determination of Kd for Cells in Suspension

  Materials
  • Adherent cells and incubation medium
  • 25‐mm glass coverslip mounted in a Leiden chamber (Medical Systems Corp.)
  • 1× low‐calcium medium (see recipe)
  • 1× high‐calcium medium (see recipe)
  • Standard medium with cyanide (see recipe)
  • Fluorescence microscope with FDIM system (Photon Technology International, Inc.)
  • Additional reagents and equipment for fluorescence measurement of intracellular [Ca2+] (see protocol 1)

Basic Protocol 2: Measurements of [Ca2+] in Plated Cells Using Fluorescence Digital Imaging Microscopy (FDIM)

  • EGTA solution (see recipe for Ca2+ calibration standards)
  • CaEGTA solution (see recipe for Ca2+ calibration standards)
  • Ca solution (see recipe for Ca2+ calibration standards)
  • 1 M CaCl 2
  • Cells incubated with probe (see protocol 1, step )
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Figures

Videos

Literature Cited

Literature Cited
   Baker, A.J., Brandes, R., Schreur, J.H.M., Camacho, S.A., and Weiner, M.W. 1994. Protein and acidosis alter calcium‐binding and fluorescence spectra of the calcium indicator Indo‐1. Biophys. J. 67:1646‐1654.
   Bassani, J.W.M., Bassani, R.A., and Bers, D.M. 1995. Calibration of Indo‐1 and resting intracellular [Ca]i in intact rabbit cardiac myocytes. Biophys. J. 68:1453‐1460.
   Brandes, R., Figueredo, V.M., Camacho, S.A., Baker, A.J., and Weiner, M.W. 1993. I. Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo‐1 fluorometry. Biophys. J. 65:1973‐1982.
   di Virgilio, F., Steinberg, T.H., and Silverstein, S.C. 1990. Inhibition of Fura‐2 sequestration and secretion with organic anion transport blockers. Cell Calcium 11:57‐62.
   Fiskum, G. 1985. Intracellular levels and distribution of Ca2+ in digitonin‐permeabilized cells. Cell Calcium 6:25‐37.
   Gores, G.J., Nieminen, A.‐L., Wray, B.E., Herman, B., and Lemasters, J.J. 1989. Intracellular pH during “chemical hypoxia” in cultured rat hepatocytes. J. Clin. Invest. 83:386‐396.
   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.
   Gunter, T.E., Restrepo, D., and Gunter, K.K. 1988. Conversion of esterified Fura‐2 and Indo‐1 to Ca2+‐sensitive forms by mitochondria. Am. J. Physiol. 255:c304‐c310.
   Gunter, T.E., Zuscik, M.J. Puzas, J.E., Gunter, K.K., and Rosier, R.N. 1990. Cytosolic free calcium concentration in avian growth plate chondrocytes. Cell Calcium 11:445‐457.
   Harkins, A.B., Kurebayashi, N., and Baylor, S. 1993. Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with Fluo‐3. Biophys. J. 65:865‐881.
   Homolya, L., Hollò, Z., Germann, U.A., Pastan, I., Gottesman, M.M., and Sarkadi, B. 1993. Fluorescent cellular indicators are extruded by the multidrug resistance protein. J. Biol. Chem. 268:21493‐21496.
   Jiang, Y. and Julian, F.J. 1997. Pacing rate, halothane, and BDM affect Fura 2 reporting of [Ca2+]i in intact rat trabeculae. Am. J. Physiol. 273:C2046‐2056.
   Kao, J.P.Y. 1994. Practical aspects of measuring [Ca2+] with fluorescent indicators. Methods Cell Biol. 40:155‐181.
   Lemasters, J.J. 1996. Confocal microscopy of single living cells. In Fluorescence Imaging Spectroscopy and Microscopy (X.F.Wang and B.Herman, eds.) pp. 157‐175. John Wiley & Sons, New York.
   Lipp, P., Lüscher, C., and Niggli, E. 1996. Photolysis of caged compounds characterized by ratiometric confocal microscopy: A new approach to homogeneously control and measure the calcium concentration in cardiac myocytes. Cell Calcium 19:255‐266.
   Moisescu, D.G. and Pusch, H. 1975. A pH‐metric method for the determination of the relative concentration of calcium to EGTA. Pfluegers Arch. 355:R122.
   Moore, E.D.W., Becker, P.L., Fogarty, K.E., Williams, D.A., and Fay, F.S. 1990. Ca2+ imaging in single living cells:Theoretical and practical issues. Cell Calcium 11:157‐179.
   Oakes, S.G., Martin, II,W.J., Lisek, C.A., and Powis, G. 1988. Incomplete hydrolysis of the calcium precursor Fura‐2 pentaacetoxymethyl ester (Fura‐2 AM) by cells. Anal. Biochem. 169:159‐166.
   Poenie, M., Alderton, J. Steinhardt, R., and Tsein, R. 1986. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science. 233:886‐889.
   Roe, M.W., Lemasters, J.J., and Herman, B. 1990. Assessment of Fura‐2 for measurements of cytosolic free calcium. Cell Calcium 11:63‐73.
   Trollinger, D.R., Cascio, W.E., and Lemasters, J.J. 1997. Selective loading of Rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes. Biochem. Biophys. Res. Commun. 236:738‐742.
   Tsien, R.Y., Pozzan, T., and Rink, T.J. 1982. Calcium homeostasis in intact lymphocytes:Cytosolic free calcium monitored with a new intracellular trapped fluorescent indicator. J. Cell Biol. 94:325‐334.
   Williams, D.A. and Fay, F.S. 1990. Intracellular calibration of the fluorescent calcium indicator Fura‐2. Cell Calcium 11:75‐83.
Key References
   Bassani, et al., 1995. See above.
  Describes how to measure Kd, Rmax, and Rmin inside of cells and the difficulties in so doing, including possible errors.
   Kao, 1994. See above.
  Excellent general reference.
   McGuigan, et al., 1991.
  Describes in detail how to make accurate calcium buffers.
   Moore, et al., 1990. See above.
  Excellent all‐around reference, especially with respect to theory.
   Roe, et al., 1990. See above.
  Good all‐around reference, especially with respect to probe loading and probe loading problems. Discusses advantages and disadvantages of in vitro and in situ calibration of probe.
   Williams and Fay, 1990. See above.
  Describes in detail how to choose the proper combination of ionophores for a particular cell type, including determining how much of each is needed.
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