Overview of Functional Cell Assays

Peter S. Rabinovitch1, J. Paul Robinson2

1 University of Washington, Seattle, Washington, 2 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.1
DOI:  10.1002/0471142956.cy0901s21
Online Posting Date:  May, 2002
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Abstract

Flow cytometry can measure a variety of functional parameters of increasing interest to cell biologists and clinicians. The distinguishing feature of these assays is that the information of interest is the functional behavior of the cell itself, rather than the phenotype. This unit discusses cell viability, oxidative metabolism, membrane potential, intracellular ionized calcium, intracellular pH, intracellular organelles, and gene reporter assays.

     
 
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Table of Contents

  • Cell Viability
  • Oxidative Metabolism
  • Membrane Potential
  • Intracellular Ionized Calcium
  • Intracellular pH
  • Intracellular Organelles
  • Gene Reporter Assays
  • Literature Cited
  • Tables
     
 
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Materials

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Figures

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Literature Cited

Literature Cited
   Carter, W.O. and Robinson, J.P. 1994. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukocyte Biol. 55:253‐258.
   Carulli, G., Minnucci, S., Vanacore, R., and Ambrogi, F. 1995. The role of flow cytometry in the study of physiopathology of neutrophilic granulocytes. Recent Prog. Med. 86:208‐216.
   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.
   Maftah, A., Huet, O., Gallet, P.F., and Ratinaud, M.H. 1993. Flow cytometry's contribution to the measurement of cell functions. Biol. Cell 78:85‐93.
   Minta, A., Kao, J.P.Y., and Tsien, R.Y. 1989. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264:8171‐8178.
   Novak, E.J. and Rabinovitch, P.S. 1994. Improved sensitivity in intracellular ionized calcium measurement using fluo‐3/Fura Red fluorescence ratios. Cytometry 17:135‐141.
   Petit, J.M., Denis‐Gay, M., and Ratinaud, M.H. 1993. Assessment of fluorochromes for cellular structure and function studies by flow cytometry. Biol. Cell 78:1‐13.
   Rabinovitch, P.S. and June, C.H. 1990. Measurement of intracellular free calcium and membrane potential. In Flow Cytometry and Cell Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 651‐668. Wiley‐Liss, New York.
   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.
   Rabinovitch, P.S., June, C.H., and Kavanagh, T.J. 1992. Measurements of cell physiology: Ionized calcium, pH and glutathione. In Clinical Flow Cytometry: Principles and Applications (K.D. Bauer, R.E. Duque, and T.V. Shankey, eds.) pp. 505‐534. Williams & Wilkins, Baltimore.
   Rijkers, G.T., Justement, L.B., Griffioen, A.W., and Cambier, J.C. 1990. Improved method for measuring intracellular Ca++ with fluo‐3. Cytometry 11:923‐927.
   Robinson, J.P., Carter, W.O., and Narayanan, P.K. 1994. Oxidative product formation analysis by flow cytometry. Methods Cell Biol. 41:437‐447.
   Rothe, G., Kellermann, W., and Valet, G. 1990. Flow cytometric parameters of neutrophil function as early indicators of sepsis‐ or trauma‐related pulmonary or cardiovascular organ failure. J. Lab. Clin. Med. 115:52‐61.
   Shapiro, H.M. 1994. Cell membrane potential analysis. Methods Cell Biol. 41:121‐113.
   Shapiro, H.M., Natale, P.J., and Kamentsky, L.A. 1979. Estimation of membrane potentials of individual lymphocytes by flow cytometry. Proc. Natl. Acad. Sci. U.S A. 76:5728‐5730.
   Tsien, R.Y. 1981. A non‐disruptive technique for loading calcium buffers and indicators into cells. Nature 290:527‐528.
   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
   Valet, G., Raffael, A., Moroder, L., Wünsch, E., and Ruhenstroth‐Bauer, G. 1981. Fast intracellular pH determination in single cells by flow‐cytometry. Naturwissenschaften 68:265‐266.
   Whitaker, J.E., Haugland, R.P., and Prendergast, F.G. 1988. Seminaphtho‐fluoresceins and‐rhodafluors: Dual fluorescence pH indicators. Biophys. J. 53:197a.
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