Measurement of Free Intracellular Calcium (Cai) in Fibroblasts

Digital Image Analysis of Fura 2 Fluorescence
  • R. W. Tucker
  • K. Meade-Cobun
  • H. Loats
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)


Measurement of intracellular calcium has long been a goal in many areas of biology. Calcium has been heralded as an intracellular messenger because of its unique structure and binding properties (Kretsingeret al., 1980), which allow Ca2+ ion to bind and activate specific regulatory molecules, such as calmodulin (Cheung, 1980). Ca2+ -activated processes have been implicated in signal transduction in metabolism, neural transmission, excitation-con traction coupling, and mitosis, and, more recently, in the stimulation of early mitogenic events in cells as diverse as fibroblasts (Boyntonet al., 1980; Chafouleaset al., 1982; Morriset al., 1984; McNeilet al., 1985; Tuckeret al., 1986) and lymphocytes (Heskethet al., l983;Gelfandet al., 1986).


Excitation Spectrum Free Calcium Digital Image Analysis Cytoplasmic Free Calcium Thick Ascend Limb Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Agard, D. A., and Sedat, J. W., 1983, Three-dimensional architecture of a polytene nucleus, Nature 302:676–681.PubMedCrossRefGoogle Scholar
  2. Boynton, A. L., Whitfield, J. R., and MacManus, J. P., 1980, Calmodulin stimulates DNA synthesis in rat liver cells, Biochem . Biophys. Res. Commun. 95:745–749.PubMedCrossRefGoogle Scholar
  3. Bright, G. R., Fisher, G. W., Rogowska, J., and Taylor, D. L., 1987, Fluorescence ratio imaging microscopy: temporal and spatial measurement of cytoplasmic pH, J. Cell Biol. 104:1019–1033.PubMedCrossRefGoogle Scholar
  4. Cerutti, P., 1988, Oxidant tumor promotors, Toxicol . Pathol. 16:73–74.Google Scholar
  5. Chafouleas, J. G., Bolton, W. E., Hidaka, H., Boyd, A. E., and Means, A. R., 1982, Calmodulin and the cell cycle: Involvement in regulation of cell-cycle progression, Cell 28:41–50.PubMedCrossRefGoogle Scholar
  6. Cheung, W. Y., 1980, Calmodulin-An introduction, in: Calcium and Cell Function ,Vol. 1 (W. Y. Cheung, ed.), Academic Press, New York, pp. 1–9.Google Scholar
  7. Connor, J. A., 1986, Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells, Proc. Natl. Acad. Sci. USA 83: 6179–6183.PubMedCrossRefGoogle Scholar
  8. Connor, J. A., Cornwall, M. C., and Williams, G. H., 1987, Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomerulosa cells measured by digital imaging techniques. J. Biol. Chem. 262:2919–2927.PubMedGoogle Scholar
  9. Cornejo, M., Guggino, S., and Guggino, W. B., 1987, Modification of calcium-activated potassium channel in cultured medullary thick ascending limb cells by N-bromoacetamide, J. Memb. Biol. 99:147–155.CrossRefGoogle Scholar
  10. Fay, F. S., and Tucker, R. W., 1985, Studies of free cytosolic calcium (Cai) in quiescent BALB/c 3T3 cells using Quin2 and Fura 2, J. Cell Biol. 101:476a.Google Scholar
  11. Gelfand, E. W., Cheung, R. K., Grinstein, S., and Mills, G. B., 1986, Characterization of the role for calcium influx in mitogen-induced triggering of human T cells. Identification of calciumdependent and calcium-independent signals, Eur. J. Immunol. 16:907–912.PubMedCrossRefGoogle Scholar
  12. 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.PubMedGoogle Scholar
  13. Hesketh, T. R., Smith, G. A., Moore, J. P., Taylor, M. V., and Metcalfe, J. C., 1983, Free cytoplasmic calcium concentration and the mitogenic stimulation of lymphocytes, J. Biol. Chem. 258:4876–4882.PubMedGoogle Scholar
  14. Kretsinger, R. H., 1980, Structure and evolution of calcium-modulated proteins, CRC Crit. Rev. Biochem. 8:114–174.CrossRefGoogle Scholar
  15. Lemasters, J. J., DiGuiseppi, J., Nieminen, A.-L., and Herman, B., 1987, Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes, Nature 325:78–81.PubMedCrossRefGoogle Scholar
  16. McNeil, P. L., McKenna, M. P., and Taylor, D. L., 1985, A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate, J. Cell. Biol. 101:372–379.PubMedCrossRefGoogle Scholar
  17. Millard, P., Gross, D., Webb, W., and Fewtrell, C., 1988, Imaging asynchronous changes in intracellular Ca2+ in individual stimulated tumor mast cells, Proc. Natl. Acad. Sci. 85:1854–1858.PubMedCrossRefGoogle Scholar
  18. Morris, J. D. H., Metcalfe, J. C., Smith, G. A., Hesketh, T. R., and Taylor, M. V., 1984, Some mitogens cause rapid increases in free calcium in fibroblasts, FEBS Lett. 169:189–193.PubMedCrossRefGoogle Scholar
  19. Nemeth, E. F. and Scarpa, A., 1986, Cytosolic Ca2+ and the regulation of secretion in parathyroid cells, FEBS Lett. 203:15–19.PubMedCrossRefGoogle Scholar
  20. Pershadsingh, H. A., Shade, D. L., Delfert, D. M., and McDonald, J. M., 1987, Chelation of intracellular calcium blocks insulin action in the adipocyte, Proc. Natl. Acad. Sci. U.S.A. 84:1025– 1029.PubMedCrossRefGoogle Scholar
  21. Poenie, N., Alderton, J., Steinhardt, R., and Tsien, R. Y., 1986, Calcium rises abruptly and briefly throughout the cell at the onset of amaphase, Science 233:886–889.PubMedCrossRefGoogle Scholar
  22. Pozzan, T. Arslan, P., Tsien, R. Y., and Rink, T. J., 1982, Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes, J. Cell Biol. 94:335–340.PubMedCrossRefGoogle Scholar
  23. Rabinovitch, P. S., June, C. H., Grossman, 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. Immol. 137:952–961.Google Scholar
  24. Ratan, R. R., Shelanski, M. L., and Maxfield, F. R., 1986, Transition from metaphase to anaphase is accompanied by local changes in cytoplasmic free calcium in Pt K2 kidney epithelial cells, Proc. Natl. Acad. Sci. USA 83:5136–5140.PubMedCrossRefGoogle Scholar
  25. Resendez, E. Jr., Attenello, J. W., Grafsky, A., Chang, C. S.,and Lee, A. S., 1985, Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes, Molec . Cell. Biol. 5:1212–1219.PubMedGoogle Scholar
  26. Sequin, C. and Hamer, D. H., 1987, Regulation in vitro of metallothionein gene binding factors, Science 235:1383–1387.CrossRefGoogle Scholar
  27. 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.PubMedGoogle Scholar
  28. Simons, J. W., Noga, S. J., Colombani, P. M., Beschorner, W. E., Coffey, D. S., and Hess, A. D., 1986, Cyclosporine A, an in vitro calmodulin antagonist, induces nuclear lobulations in human T cell lymphocytes and monocytes, J. Cell Biol. 102:145–150.PubMedCrossRefGoogle Scholar
  29. Tucker, R. W., and Loats, H., 1987, Analysis of intracellular free cytosolic calcium in human fibroblasts using Fura-2 and digital image analysis with a microcomputer, Ann. N.Y. Acad. Sci. 494:283–286.CrossRefGoogle Scholar
  30. Tucker, R. W., Snowdowne, K. W., and Borle, A. B., 1986, Cytosolic free calcium and DNA synthesis in BALB/c 3T3 cells: aequorin luminescence studies, Eur. J. Cell Biol. 41:347–351.PubMedGoogle Scholar
  31. Tsien, R. Y., Rink, T. J., and Poenie, M., 1985, Measurement of cytosolic free Ca2+ in individual small cells using fluorescent microscopy with dual excitation wavelengths, Cell Calcium 6:145–157.PubMedCrossRefGoogle Scholar
  32. Wier, W. G., Cannell, M. B., Berlin, J. R., Marban, E., and Leaderer, W. J., 1987, Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by Fura-2, Science 235:325–328.PubMedCrossRefGoogle Scholar
  33. White, B. A., 1985, Evidence for a role of calmodulin with regulation of prolactin gene expression, J. Biol. Chem. 260:1213–1217.PubMedGoogle Scholar
  34. Williams, D. A., Fogarty, K. E., Tsien, R. Y., and Fay, F. S., 1985, Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2, Nature 318:558–561.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • R. W. Tucker
    • 1
  • K. Meade-Cobun
    • 1
  • H. Loats
    • 2
  1. 1.Johns Hopkins Oncology CenterBaltimoreUSA
  2. 2.Loats Associates, Inc.WestminsterUSA

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