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

  • 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

  • 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

  • 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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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‐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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