Flow Cytometric Measurement of Intracellular pH

Sue Chow1, David Hedley1

1 Ontario Cancer Institute and Princess Margaret Hospital, Toronto, Ontario, Canada
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.3
DOI:  10.1002/0471142956.cy0903s14
Online Posting Date:  May, 2001
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Abstract

A number of fundamentally important biological processes, such as cell signaling and the initiation of mitosis, are accompanied by a change in intracellular pH. Flow cytometric measurement of pH is a generally straightforward procedure that can be done with any instrument equipped with a 488‐nm argon laser. The overall approach is similar to that for calcium: generation of a calibration curve by imparting known changes in pH and interpolation of the test sample pH. This unit presents the traditional calibration method using high‐potassium buffers and the proton ionophore nigericin and a more recently developed technique, the pseudo null method, which involves resuspension of cells in defined mixtures of weak acids and weak bases.

     
 
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Table of Contents

  • Basic Protocol 1: pH Measurements with SNARF‐1 Using Nigericin Calibration
  • Alternate Protocol 1: pH Measurement with SNARF‐1 Using Pseudo Null Calibration
  • Alternate Protocol 2: pH Measurement with BCECF
  • Support Protocol 1: Preparation of High‐Potassium Calibration Buffer Series
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: pH Measurements with SNARF‐1 Using Nigericin Calibration

  Materials
  • Cell sample
  • PBS ( appendix 2A)
  • 2 mM carboxy SNARF‐1 AM (see recipe)
  • High‐potassium calibration buffers (see protocol 4Support Protocol 1)
  • 1 mg/ml nigericin (see recipe)
  • 12 × 75–mm polypropylene or polystyrene tubes (Falcon) as required by flow cytometer
  • Flow cytometer with 488‐nm argon laser and ratio parameter, if available
  • Bandpass filters centered at or around 580 and 640 nm

Alternate Protocol 1: pH Measurement with SNARF‐1 Using Pseudo Null Calibration

  • 10 mM HEPES buffer (see recipe)
  • HDFBS: 10 mM HEPES buffer supplemented with 10% dialyzed fetal bovine serum ( FBS)
  • 6× pseudo null calibration solutions (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Boyer, M.J. and Hedley, D.W. 1994. Measurement of intracellular pH. Methods Cell Biol. 41:135‐149.
   Busa, W.B. and Nuccitelli, R. 1984. Metabolic regulation via intracellular pH. Am. J. Physiol. 246:R409‐R438.
   Chow, S., Hedley, D.W., and Tannock, I.F. 1996. Flow cytometric calibration of intracellular pH measurements in viable cells using mixtures of weak acids and bases. Cytometry 24:360‐367.
   Eisner, D.A., Kenning, N.A., O'Neill, S.C., Pocock, G., Richards, C.D., and Valdeolmillos, M.A. 1989. A novel method for absolute calibration of intracellular pH indicators. Pflügers Archiv.Eur. J. Physiol. 413:553‐558.
   Grinstein, S., Rotin, D., and Mason, M.J. 1989. Na+/H+ exchange and growth factor–induced cytosolic pH changes. Role in cellular proliferation. Biochim. Biophys. Acta 988:73‐367.
   Lagarde, A.E. and Pouyssegur, J.M. 1986. Mini Review: The Na+:H+ antiport in cancer. Cancer Biochem. Biophys. 9:1‐14.
   Musgrove, E.A., Rugg, C.A., and Hedley, D.W. 1987. Flow cytometric measurement of intracellular pH: A critical evaluation of available fluorochromes. Cytometry 7:347‐355.
   Pressman, B.C. and de Guzman, N.T. 1977. Biological applications and evolutionary origins of ionophores. Adv. Exp. Med. Biol. 84:285‐300.
   Sutherland, R. 1986. Importance of critical metabolites and cellular interactions in the biology of microregions of tumors. Cancer 58:1668‐1680.
   Szatkowski, M.S. and Thomas, R.C. 1986. New method for calibrating pHi from accurately measured changes in pHi induced by a weak acid and base. Pflügers Archiv. Eur. J. Physiol. 407:59‐63.
   Wieder, E.D., Hang, H., and Fox, M.H. 1993. Measurement of intracellular pH using flow cytometry with carboxy‐SNARF‐1. Cytometry 14:916‐921.
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