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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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

  • 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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Materials

Basic Protocol 1: 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 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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Figures

Videos

Literature Cited

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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 http://www.probes.com/handbook/
   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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