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Measurement of Intracellular Ions by Flow Cytometry

Carl H. June1,  Jonni S. Moore1

1University 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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Abstract

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

  • Unit Introduction
  • Basic Protocol: 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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Materials

Basic Protocol: Use of Indo-1 AM and Flow Cytometry to Measure Cellular Calcium Concentration

 Materials
  • 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 PBS (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 (Support Protocol 1), 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

 Additional Materials (also see Basic Protocol)
  • 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

 Additional Materials (also see Basic Protocol)
  • 2 mg/ml Fluo-3 acetoxymethyl ester (Fluo-3 AM; see recipe)
  • HBSS or PBS (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

 Additional Materials (also see Basic Protocol)
  • Calcium calibration buffer concentrates (Molecular Probes): zero calcium (100 mM K2H2EGTA) and 100 mM calcium (100 mM K2CaEGTA)
  • 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

 Additional Materials (also see Basic Protocol)
  • HBSS (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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Figures

  • Figure 5.5.1
    Example of in situ calibration of Indo-1 ratio shifts. Human T cells were loaded with Indo-1 and electrochemical gradients disabled as described in the text. The cells were suspended in a series of calcium buffers ranging from 6 nM to 22 µM and steady-state Indo-1 fluorescence ratios determined. The ratio of the 22-µM sample was off-scale and was greater than channel 1024.

    View Image
  • Figure 5.5.2
    Effects of T cell receptor stimulation on CD4 cell ionized calcium concentration ([Ca2+]i). Peripheral blood lymphocytes (PBL) were loaded with Indo-1 AM and stained with PE-anti-CD8. The cells were maintained at 37°C; after obtaining a baseline for ~1 min, anti-CD3 MAb was added during the gap in analysis. (A) The Indo-1 ratio of 395 nm/500 nm fluorescence emission was calculated and the value for each cell displayed on the y axis versus time on the x axis. The results are displayed as a “dot plot” on a 100 × 100 pixel grid, where the number of cells per pixel is represented by increasing shades of gray. Changes in [Ca2+]i in the CD4 subset of T cells are depicted by setting electronic gates on the Indo-1 fluorescence derived from the PE-negative cells. (B) The mean response of the data from panel A plotted versus time.

    View Image

Literature Cited

Literature Cited
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    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.
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    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.
    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.

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 http://www.probes.com/handbook/

    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.

Excellent overviews of calibration strategies and probes.

    Takahashi, A., Camacho, P., Lechleiter, J.D., and Herman, B. 1999. Measurement of intracellular calcium. Physiol. Rev. 79:1089-1125.

Comprehensive overview of optically and non-optically based intracellular calcium assays.

     
 
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