Measurement of Intracellular Ions by Flow Cytometry

Carl H. June1, Jonni S. Moore1

1 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.5
DOI:  10.1002/0471142735.im0505s64
Online Posting Date:  December, 2004
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The recent development of a number of new fluorescent probes makes it possible to measure the concentrations of various intracellular free ions in single living cells. Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH). This unit describes flow cytometric protocols using the dyes Indo‐1 AM, Fluo‐3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer.

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Table of Contents

  • Basic Protocol 1: Use of Indo‐1 AM and Flow Cytometry to Measure Cellular Calcium Concentration
  • Alternate Protocol 1: Simultaneous use of Fluo‐3 and Fura Red AM Fluorescence Ratios for Flow Cytometric Calcium Measurement
  • Alternate Protocol 2: Use of a Spectrofluorimeter to Determine [Ca2+]i
  • Support Protocol 1: Use of Calcium/EGTA Buffers to Calibrate Flow Cytometric Calcium Measurements
  • Support Protocol 2: Use of Pluronic Detergent F‐127 to Load Cells with Indo‐1 AM, Fluo‐3 AM, or Fluo‐3 AM and Fura Red AM
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Use of Indo‐1 AM and Flow Cytometry to Measure Cellular Calcium Concentration

  • Murine splenic lymphocytes (unit 3.1) or human peripheral blood lymphocytes (unit 7.1)
  • Cell loading medium (see recipe)
  • 100 mM probenecid (see recipe)
  • 2 mg/ml Indo‐1 pentaacetoxymethyl ester (Indo‐1 AM; see recipe)
  • 1 mg/ml ionomycin (see recipe)
  • Dimethyl sulfoxide (DMSO; Sigma) or 10% (v/v) bleach in water
  • Saline: 0.85% (w/v) NaCl or recipePBS ( appendix 2A)
  • Beckman TJ‐6 rotor (or equivalent)
  • 12 × 75–mm polypropylene tubes (Falcon)
  • 30° or 37°C water bath
  • Fluorescence microscope
  • Flow cytometer with UV light source and heated sample chamber (e.g., Becton Dickinson, Coulter, or Ortho), and software for kinetic and ratiometric analysis (Phoenix Flow Systems)
  • Additional reagents and equipment for flow cytometry (unit 5.4), calibration of calcium measurements ( protocol 4), and analysis of data (unit 5.2)

Alternate Protocol 1: Simultaneous use of Fluo‐3 and Fura Red AM Fluorescence Ratios for Flow Cytometric Calcium Measurement

  • 10 mg/ml Fluo‐3 acetoxymethyl ester (Fluo‐3 AM; see recipe)
  • 10 mg/ml Fura Red AM acetoxymethyl ester (Fura Red AM; see recipe)
  • Appropriately labeled antibody (optional)

Alternate Protocol 2: Use of a Spectrofluorimeter to Determine [Ca2+]i

  • 2 mg/ml Fluo‐3 acetoxymethyl ester (Fluo‐3 AM; see recipe)
  • recipeHBSS or recipePBS ( appendix 2A) containing 1 mM Ca2+ but without phenol red or FBS
  • Anti‐fluorescein rabbit IgG (H + L) fraction (Molecular Probes; optional)
  • 5% (v/v) Triton X‐100
  • 100 mM EGTA, pH 8
  • 1 M Tris base, pH 9.4, in water
  • Quartz cuvette or UV‐transparent plastic disposable cuvettes
  • Spectrofluorimeter equipped with stirrer and temperature‐controlled cuvette

Support Protocol 1: Use of Calcium/EGTA Buffers to Calibrate Flow Cytometric Calcium Measurements

  • Calcium calibration buffer concentrates (Molecular Probes): zero calcium (100 mM K 2H 2EGTA) and 100 mM calcium (100 mM K 2CaEGTA)
  • Poisoned DPBS (see recipe)
  • Indo‐1 or Fluo‐3 salt (not ester; Molecular Probes; optional)

Support Protocol 2: Use of Pluronic Detergent F‐127 to Load Cells with Indo‐1 AM, Fluo‐3 AM, or Fluo‐3 AM and Fura Red AM

  • recipeHBSS ( appendix 2A) containing 1% (v/v) FBS
  • 50‐µg vial Indo‐1 AM pentaacetoxymethyl ester (Indo‐1 AM; Molecular Probes)
  • 20% (w/v) pluronic F‐127 (Molecular Probes) in DMSO; warm at 37°C until dissolved
  • FBS (heat inactivated 1 hr at 56°C)
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Literature Cited

   Abe, R., Ishida, Y., Yui, K., Katsumata, M., and Chused, T.M. 1992. T cell receptor‐mediated recognition of self‐ligand induces signaling in immature thymocytes before negative selection. J. Exp. Med. 176:459‐468.
   Alexander, R.B., Bolton, E.S., Koenig, S., Jones, G.M., Topalian, S.L., June, C.H., and Rosenberg, S.A. 1992. Detection of antigen‐specific T lymphocytes by determination of intracellular calcium concentration using flow cytometry. J. Immunol. Methods 148:131‐141.
   Allbritton, N.L. and Meyer, T. 1993. Localized calcium spikes and propagating calcium waves. Cell Calcium 14:691‐697.
   Arslan, P., Di Virgilio, F., Beltrame, M., Tsien, R.Y., and Pozzan, T. 1985. Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane‐permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+. J. Biol. Chem. 260:2719‐2725.
   Baus, E., Urbain, J., Leo, O., and Andris, F. 1994. Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo‐3 and an organic‐anion transport inhibitor. J. Immunol. Methods 173:41‐47.
   Bers, D.M., Patton, C.W., and Nuccitelli, R. 1994. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 40:3‐29.
   Blinks, J.R., Wier, W.G., Hess, P., and Prendergast, F.G. 1982. Measurement of Ca2+ concentrations in living cells. Prog. Biophys. Mol. Biol. 40:1‐114.
   Chien, M.M., Zahradka, K.E., Newell, M.K., and Freed, J.H. 1999. Fas‐induced B cell apoptosis requires an increase in free cytosolic magnesium as an early event. J. Biol. Chem. 274:7059‐7066.
   Chused, T.M., Wilson, H.A., Greenblatt, D., Ishida, Y. L., Edison, J., Tsien, R.Y., and Finkelman, F.D. 1987. Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti‐IgM and anti‐IgD. Cytometry 8:396‐404.
   Cobbold, P.H. and Rink, T.J. 1987. Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem. J. 248:313‐328.
   Dewitt, S., Laffafian, I., Morris, M.R., and Hallett, M.B. 2003. Cytosolic Ca2+ measurement and imaging in inflammatory cells. Methods Mol. Biol. 225:47‐59.
   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.
   Ganz, M.B., Rasmussen, J., Bollag, W.B., and Rasmussen, H. 1990. Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2. FASEB J. 4:1638‐1644.
   Gee, K.R., Archer, E.A., Lapham, L.A., Leonard, M.E., Zhou, Z.L., Bingham, J., and Diwu, Z. 2000a. New ratiometric fluorescent calcium indicators with moderately attenuated binding affinities. Bioorg. Med. Chem. Lett. 10:1515‐1518.
   Gee, K.R., Brown, K.A., Chen, W.N., Bishop‐Stewart, J., Gray, D., and Johnson, I. 2000b. Chemical and physiological characterization of fluo‐4 Ca(2+)‐indicator dyes. Cell Calcium 27:97‐106.
   Gelfand, E.W. and Cheung, R.K. 1990. Dissociation of unidirectional influx of external Ca2+ and release from internal stores in activated human T lymphocytes. Eur. J. Immunol. 20:1237‐1241.
   Gelfand, E.W., MacDougall, S.L., Cheung, R.K., and Grinstein, S. 1989. Independent regulation of Ca2+ entry and release from internal stores in activated B cells. J. Exp. Med. 170:315‐320.
   Goldsmith, M.A. and Weiss, A. 1987. Isolation and characterization of a T‐lymphocyte somatic mutant with altered signal transduction by the antigen receptor. Proc. Natl. Acad. Sci. U.S.A. 84:6879‐6883.
   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.
   Hadley, R.W., Kirby, M.S., Lederer, W.J., and Kao, J.P. 1993. Does the use of DM‐nitrophen, nitr‐5, or diazo‐2 interfere with the measurement of Indo‐1 fluorescence? Biophys. J. 65:2537‐2546.
   Harkins, A.B., Kurebayashi, N., and Baylor, S.M. 1993. Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo‐3. Biophys. J. 65:865‐881.
   Ishida, Y. and Chused, T.M. 1988. Heterogeneity of lymphocyte calcium metabolism is caused by T cell‐specific calcium‐sensitive potassium channel and sensitivity of the calcium ATPase pump to membrane potential. J. Exp. Med. 168:839‐852.
   Kachel, V., Kempski, O., Peters, J., and Schodel, F. 1990. A method for calibration of flow cytometric wavelength shift fluorescence measurements. Cytometry 11:913‐915.
   Lanza, F., Beretz, A., Kubina, M., and Cazenave, J.P. 1987. Increased aggregation and secretion responses of human platelets when loaded with the calcium fluorescent probes quin2 and fura‐2. Thromb. Haemost. 58:737‐743.
   Li, Q., Altschuld, R.A., and Stokes, B.T. 1987. Quantitation of intracellular free calcium in single adult cardiomyocytes by fura‐2 fluorescence microscopy: Calibration of fura‐2 ratios. Biochem. Biophys. Res. Commun. 147:120‐126.
   Liddle, R.A., Misukonis, M.A., Pacy, L., and Balber, A.E. 1992. Cholecystokinin cells purified by fluorescence‐activated cell sorting respond to monitor peptide with an increase in intracellular calcium. Proc. Natl. Acad. Sci. U.S.A. 89:5147‐5151.
   Lipp, P. and Niggli, E. 1993. Ratiometric confocal Ca2+‐measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium 14:359‐372.
   Luckhoff, A. 1986. Measuring cytosolic free calcium concentration in endothelial cells with indo‐1: The pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths. Cell Calcium 7:233‐248.
   Lupu‐Meiri, M., Beit‐Or, A., Christensen, S.B., and Oron, Y. 1993. Calcium entry in Xenopus oocytes: Effects of inositol trisphosphate, thapsigargin and DMSO. Cell Calcium 14:101‐110.
   Malgaroli, A., Milani, D., Meldolesi, J., and Pozzan, T. 1987. Fura‐2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells. J. Cell Biol. 105:2145‐2155.
   Merritt, J.E., McCarthy, S.A., Davies, M.P., and Moores, K.E. 1990. Use of fluo‐3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+. Biochem. J. 269:513‐519.
   Miller, D.J. and Smith, G.L. 1984. EGTA purity and the buffering of calcium ions in physiological solutions. Am. J. Physiol. 246:C160‐C166.
   Minta, A., Kao, J.P., and Tsien, R.Y. 1989. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264:8171‐8178.
   Monteith, G.R. 2000. Seeing is believing: Recent trends in the measurement of Ca2+ in subcellular domains and intracellular organelles. Immunol. Cell Biol. 78:403‐407.
   Negulescu, P.A. and Machen, T.E. 1990. Intracellular ion activites and membrane transport in parietal cells measured with fluorescent dyes. Methods Enzymol. 192:38‐81.
   Novak, E.J. and Rabinovitch, P.S. 1994. Improved sensitivity in intracellular ionized calcium measurement using fluo‐3/fura red fluorescence ratios. Cytometry 17:135.
   Oiki, S., Yamamoto, T., and Okada, Y. 1994. Apparent stability constants and purity of Ca‐ chelating agents evaluated using Ca‐selective electrodes by the double‐log optimization method. Cell Calcium 15:209‐216.
   Osipchuk, Y. and Cahalan, M. 1992. Cell‐to‐cell spread of calcium signals mediated by ATP receptors in mast cells. Nature 359:241‐244.
   Owen, C.S. and Shuler, R.L. 1989. Spectral evidence for noncalcium interactions of intracellular Indo‐1. Biochem. Biophys. Res. Commun. 163:328‐333.
   Pesco, J., Salmon, J.M., Vigo, J., and Viallet, P. 2001. Mag‐indo1 affinity for Ca(2+), compartmentalization and binding to proteins: the challenge of measuring Mg(2+) concentrations in living cells. Anal. Biochem. 290:221‐231.
   Pethig, R., Kuhn, M., Payne, R., Adler, E., Chen, T.H., and Jaffe, L.F. 1989. On the dissociation constants of BAPTA‐type calcium buffers. Cell Calcium 10:491‐498.
   Poenie, M. 1990. Alteration of intracellular fura‐2 fluorescence by viscosity: A simple correction. Cell Calcium 11:85‐91.
   Poenie, M., Alderton, J., Steinhardt, R., and Tsien, R. 1986. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science 233:886‐889.
   Rabinovitch, P.S., June, C.H., Grossmann, A., and Ledbetter, J.A. 1986. Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti‐CD3. Simultaneous use of indo‐1 and immunofluorescence with flow cytometry. J. Immunol. 137:952‐961.
   Roe, M.W., Lemasters, J.J., and Herman, B. 1990. Assessment of fura‐2 for measurements of cytosolic free calcium. Cell Calcium 11:63‐72.
   Scanlon, M., Williams, D.A., and Fay, F.S. 1987. A Ca2+‐insensitive form of fura‐2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement. J. Biol. Chem. 262:6308‐6312.
   Schild, D., Jung, A., and Schultens, H.A. 1994. Localization of calcium entry through calcium channels in olfactory receptor neurones using a laser scanning microscope and the calcium indicator dyes Fluo‐3 and Fura‐red. Cell Calcium 15:341‐348.
   Steinberg, S.F., Bilezikian, J.P., and Al‐Awqati, Q. 1987. Fura‐2 fluorescence is localized to mitochondria in endothelial cells. Am. J. Physiol. 253:C744‐C747.
   Szollosi, J., Feuerstein, B.G., Hyun, W.C., Das, M.K., and Marton, L.J. 1991. Attachment of A172 human glioblastoma cells affects calcium signalling: A comparison of image cytometry, flow cytometry, and spectrofluorometry. Cytometry 12:707‐716.
   Thomas, A.P. and Delaville, F. 1991. The use of fluorescent indicators for measurements of cytosolic‐free calcium concentration in cell populations and single cells. In Cellular Calcium: A Practical Approach (J.G. McCormack, and P.H. Cobbold, eds.) pp. 1‐54. Oxford University Press, New York.
   Tsien, R.Y. 1980. New calcium indicators and buffers with high selectivity against magnesium and protons: Design, synthesis, and properties of prototype structures. Biochemistry 19:2396‐2404.
   Tsien, R.Y. 1989. Fluorescent indicators of ion concentrations. Methods Cell Biol. 30:127‐136.
   Tsien, R.Y. 1992. Intracellular signal transduction in four dimensions: From molecular design to physiology. Am. J. Physiol. 263:C723‐C728.
   Tsien, R.Y., Pozzan, T., and Rink, T.J. 1982. Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol. 94:325‐334.
   Vandenberghe, P.A. and Ceuppens, J.L. 1990. Flow cytometric measurement of cytoplasmic free calcium in human peripheral blood T lymphocytes with fluo‐3, a new fluorescent calcium indicator. J. Immunol. Methods 127:197‐204.
   Van Graft, M., Kraan, Y.M., Segers, I.M., Radosevic, K., De Grooth, B.G., and Greve, J. 1993. Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process. Cytometry 14:257‐264.
   Williams, D.A. and Fay, F.S. 1990. Intracellular calibration of the fluorescent calcium indicator Fura‐2. Cell Calcium 11:75‐83.
Key References
   Bers et al., 1994. See above.
  A wealth of information on calcium probes, calcium buffers, and calcium ionophores. The most up‐to‐date version of Haughland's handbook is found at
   Haugland, R.P. 2003. Indicators for Ca2+, Mg2+, Zn2+ and other metal ions. In Handbook of Fluorescent Probes and Research Products, 9th ed. (K.D. Larison, ed.), Chapter 20. Molecular Probes, Inc. Eugene, Oreg.
  Excellent overviews of calibration strategies and probes.
   Pozzan, T., Mongillo, M., and Rudolf, R. 2003. The Theodore Bucher lecture: Investigating signal transduction with genetically encoded fluorescent probes. Eur. J. Biochem. 270:2343‐2352.
  Comprehensive overview of optically and non‐optically based intracellular calcium assays.
   Takahashi, A., Camacho, P., Lechleiter, J.D., and Herman, B. 1999. Measurement of intracellular calcium. Physiol. Rev. 79:1089‐1125.
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