Measurement of Intracellular Ions by Flow Cytometry

Avery D. Posey,1, Omkar U. Kawalekar1, Carl H. June1

1 Abramson Family Cancer Research Institute, and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.8
DOI:  10.1002/0471142956.cy0908s72
Online Posting Date:  April, 2015
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Abstract

Using flow cytometry, single‐cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation. © 2015 by John Wiley & Sons, Inc.

Keywords: calcium analysis; ion analysis; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Use of Indo‐1 and Flow Cytometry to Measure Cellular Calcium Concentration
  • Alternate Protocol 1: Flow Cytometric Approaches to Measure High Cellular Calcium Concentrations
  • Alternate Protocol 2: Simultaneous Use of Visual Light Excitable Calcium Indicators and Fura Red Fluorescence Ratios for Flow Cytometric Calcium Measurement
  • Alternate Protocol 3: 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 or Fluo‐3 or Fluo‐3 and Fura Red
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

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

  Materials
  • Murine splenic lymphocytes or human peripheral blood lymphocytes
  • Cell loading medium (HBSS or similar, 1 mM Ca2+, 1 mM Mg2+, 1% FBS)
  • 2 mg/ml Indo‐1 acetoxymethyl ester (Indo‐1 AM; see )
  • 100 mM probenecid (see )
  • 1 mg/ml ionomycin (see )
  • Dimethyl sulfoxide (DMSO; Sigma) or 10% bleach in water
  • Saline or phosphate‐buffered saline (PBS)
  • Beckman TJ‐6 rotor (or equivalent)
  • 12 × 75–mm polypropylene tubes (BD Biosciences, cat. no. 2063)
  • 30° or 37°C water bath
  • Fluorescence microscope
  • Flow cytometer with UV light source and heated sample chamber (e.g., Becton Dickinson LSRFortessa), and software for kinetic and ratiometric analysis (e.g., FlowJo)

Alternate Protocol 1: Flow Cytometric Approaches to Measure High Cellular Calcium Concentrations

  Materials
  • 2 mg/ml Indo‐5 F acetoxymethyl ester (Indo‐5 F AM; AnaSpec, cat. no. 84051)
  • 2 mg/ml Mag‐Indo‐1 acetoxymethyl ester (Mag‐Indo‐1 AM; Life Technologies, cat. no. M‐1295)

Alternate Protocol 2: Simultaneous Use of Visual Light Excitable Calcium Indicators and Fura Red Fluorescence Ratios for Flow Cytometric Calcium Measurement

  Additional Materials (also see the protocol 1Basic Protocol)
  • 10 mg/ml Fluo‐3 (Life Technologies, cat. no. F1241) or Fluo‐4 (Life Technologies, cat. no. F23917) or eFluor514 Calcium Sensor Dye (Affymetrix, cat. no. 65‐0859) or Rhod‐2 (Life Technologies, cat. no. R1245 MP)
  • 10 mg/ml Fura Red acetoxymethyl ester (Fura Red AM; see )
  • Appropriately labeled antibody

Alternate Protocol 3: Use of a Spectrofluorimeter To Determine [Ca2+]i

  Additional Materials (also see the protocol 1Basic Protocol and Support Protocols protocol 51 and protocol 62)
  • Hanks balanced salt solution (HBSS) or phosphate‐buffered saline (PBS) containing 1 mM Ca2+ without phenol red and FBS
  • Anti‐fluorescein rabbit IgG (H + L) fraction (Life Technologies, cat. no. A‐889)
  • 5% (v/v) Triton X‐100
  • 100 mM EGTA, pH 8
  • 1 M Tris base, pH 9.4, in water
  • 1.5‐microcentriuge tubes
  • 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

  Materials
  • Calcium Calibration Buffer Kit #1, zero, and 10 mM CaEGTA (10 mM K 2EGTA and 10 mM CaEGTA, Life Technologies, cat. no. C‐3008MP)
  • Phosphate‐buffered saline (PBS) with 20 mM HEPES, pH 7.20, without calcium or magnesium containing the following cellular poisons (use care, as these are highly toxic reagents):
    • Ionomycin 1 mg/ml stock solution in dimethyl sulfoxide (DMSO) to 3 μg/ml final
    • Nigericin 10 mg/ml stock solution in methanol to 2.0 μg/ml final
    • Carbonyl cyanide m‐chlorophenylhydrazone (CCCP) 1 mM stock solution in dimethyl sulfoxide (DMSO) at 10 μM final
    • 2‐deoxyglucose 1 M stock solution in water, 40 mM final
    • Sodium azide 3 M stock solution in water, 60 mM final
  • 37°C incubator
  • Additional reagents and equipment for loading mouse or human lymphocytes with Indo−1, or Fluo−3 plus Fura Red in PBS containing poisons (see Chused et al., )

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

  Additional Materials (also see the protocol 1Basic Protocol)
  • 20% (w/v) pluronic F‐127 in DMSO (Life Technologies, cat. no. P‐3000 MP; alternatively, use PowerLoad Concentrate, cat. no. P10020)
  • 100 mM probenecid (Life Technologies, cat. no. P36400), optional
  • Fetal bovine serum (FBS; heat‐inactivated 1 hr, 56°C)
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Figures

Videos

Literature Cited

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.
  Adams, S.R., Lev‐Ram, V., and Tsien, R.Y. 1997. A new caged Ca2+, azid‐1, is far more photosensitive than nitrobenzyl‐based chelators. Chem. Biol. 4:867‐878.
  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.
  Allen, G.J., Chu, S.P., Harrington, C.L., Schumacher, K., Hoffmann, T., Tang, Y.Y., Grill, E., and Schroeder, J.I. 2001. A defined range of guard cell calcium oscillation parameters encodes stomatal movements. Nature 411:1053‐1057.
  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. 260:2719‐2727.
  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.
  Berridge, M. 2012. Calcium signalling remodelling and disease. Biochem. Soc. Trans. 40:297‐309.
  Bers, D.M., Patton, C.W., and Nuccitelli, R. 2010. A practical guide to the preparation of Ca(2+) buffers. Methods Cell Biol. 99:1‐26.
  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., Edison, L.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., 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.
  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.
  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 Ca(2+)‐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.
  McGuigan, J.A., Kay, J.W., Elder, H.Y., and Luthi, D. 2007. Comparison between measured and calculated ionised concentrations in Mg2+ /ATP, Mg2+ /EDTA and Ca2+ /EGTA buffers; influence of changes in temperature, pH and pipetting errors on the ionised concentrations. Magnes. Res. 20:72‐81.
  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:C160‐166.
  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 activities 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 flow cytometric intracellular ionized calcium measurement using fluo‐3/Fura Red fluorescence ratios. Cytometry 17:135‐141.
  Oh‐hora, M., Komatsu, N., Pishyareh, M., Feske, S., Hori, S., Taniguchi, M., Rao, A., and Takayanagi, H. 2013. Agonist‐selected T cell development requires strong T cell receptor signaling and store‐operated calcium entry. Immunity 38:881‐895.
  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 non‐calcium 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 11:63‐73.
  Rutter, G.A., Burnett, P., Rizzuto, R., Brini, M., Murgia, M., Pozzan, T., Tavare, J.M., and Denton, R.M. 1996. Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: Significance for the regulation of pyruvate dehydrogenase activity. Proc. Natl. Acad. Sci. U.S.A. 93:5489‐5494.
  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.
  Takahashi, A., Camacho, P., Lechleiter, J.D., and Herman, B. 1999. Measurement of intracellular calcium. Physiol. Rev. 79:1089‐1125.
  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. Oxford University Press, New York.
  Tsien, R.Y. 1989. Fluorescent indicators of ion concentrations. Methods Cell Biol. 30:127‐156.
  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.
  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.
  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 127:197‐205.
  Vines, A., McBean, G.J., and Blanco‐Fernandez, A. 2010. A flow‐cytometric method for continuous measurement of intracellular Ca(2+) concentration. Cytometry A 77:1091‐1097.
  Williams, D.A. and Fay, F.S. 1990. Intracellular calibration of the fluorescent calcium indicator Fura‐2. Cell Calcium 11:75‐83.
  Zucker, R.S. 1992. Effects of photolabile calcium chelators on fluorescent calcium indicators. Cell Calcium 13:29‐40.
Key References
  Life Technologies. 2010. Chapter 19. Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions. In The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies. (eds Iain Johnson and Michelle T. Z. Spence), pp. 828‐881. Life Technologies, Inc. Eugene, Oregon. doi: 10.1134/S0006297911110101.ch19.
  A wealth of information on calcium probes, calcium buffers, and calcium ionophores. The most up‐to‐date information to the handbook is found at http://www.lifetechnologies.com/us/en/home/references/molecular‐probes‐the‐handbook.html.
  Bers and Nuccitelli, 2010. See above.
  Great overview of Ca2+ buffers and chelators as well as the effects of pH, temperature, and ionic strength on KCa.
  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 et al., 1999. See above.
  Valuable resource for flow cytometric and microscopic measurement of intracellular calcium, complementary to this protocol.
  June, C.H., Ledbetter, J.A., Rabinovitch, P.S., Martin, P.J., Beatty, P.G., and Hansen, J.A. 1986. Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes. J. Clin. Invest. 77:1224‐1232.
  First description of single‐cell calcium analysis using flow cytometry with ratiometric probe.
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