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Physiological Indicators of Cell Function

  • Michael J. Ignatius
  • Jeffrey T. Hung
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 356)

Abstract

Successful high content screening (HCS) assays place large demands on the cell-based reagents used in their development and deployment. Fortunately, there is a wide range of fluorescent physiological indicators from which to choose that are continually increasing in size and variety. Ideal fluorescent reagents for cell-based assays exhibit optimal selectivity, signal intensity, and cell solubility, yet will be easily incorporated into assays across multiple detection platforms. The repertoire of existing fluorogenic and color changing dyes that indicate physiological changes in cells for live cell kinetic and fixed end-point assays are surveyed as well as newly developed reagents for the next generation of HCS assays.

Key Words

Apoptosis assays calcium cell counts cell biology drug discovery expression reporters fluorescence high content screening high content analysis HTS high-throughput screening imaging membrane voltage microplate organelle pathway analysis 

References

  1. 1.
    Comley, J. (2005) High content screening emerging importance of novel reagents/probes and pathway analysis. Drug Discov. World Summer, 31–53.Google Scholar
  2. 2.
    Taylor, D. L. and Giuliano, K. A. (2005) Multiplexed high content screening assays create a systems cell biology approach to drug discovery. Drug Discov. Today: Technol. 2, 149–154.CrossRefGoogle Scholar
  3. 3.
    Hertzberg, R. P. and Pope, A. J. (2000) High-throughput screening: new technology for the 21st century. Chem. Biol. 4, 445–451.Google Scholar
  4. 4.
    Conrad, C., Erfle, H., Warnat, P., et al. (2004) Automatic identification of subcellular phenotypes on human cell arrays. Genome Res. 14, 1130–1136.CrossRefGoogle Scholar
  5. 5.
    Kain, S. R. (1999) Green fluorescent protein (GFP): applications in cell-based assays for drug discovery. DDT 4, 304–312.Google Scholar
  6. 6.
    Oakley, R. H., Hudson, C. C., Cruickshank, R. D., et al. (2002) The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors. Assay Drug Dev. Technol. 1, 21–30.CrossRefGoogle Scholar
  7. 7.
    Giuliano, K. A., Haskins, J. R., and Taylor, D. L. (2003) Advances in high content screening for drug discovery. Assay Drug Dev. Technol. 1, 565–577.CrossRefGoogle Scholar
  8. 8.
    Vakkila, J., DeMarco, R. A., and Lotze, M. T. (2004) Imaging analysis of STAT1 and NF-κB translocation in dendritic cells at the single cell level. J. Immunol. Methods 294, 123–134.CrossRefGoogle Scholar
  9. 9.
    Rao, A., Luo, C., and Hogan, P. G. (1997) Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15, 707–747.CrossRefGoogle Scholar
  10. 10.
    Crabtree, G. R. and Olson, E. N. (2002) NFAT signaling: choreographing the social lives of cells. Cell 109, S67–S69.CrossRefGoogle Scholar
  11. 11.
    Ashcroft, F. M. (ed) (2000) Ion Channels and Disease. Academic, San Diego, CA.Google Scholar
  12. 12.
    Doyle, J. L. and Stubbs, L. (1998) Ataxia, arrhythmia and ion-channel gene defects. Trends Genet. 14, 92–98.CrossRefGoogle Scholar
  13. 13.
    Marks, A. R. (1997) Intracellular calcium-release channels: regulators of cell life and death. Am. J. Physiol. 272, H597–H605.Google Scholar
  14. 14.
    Kuriyama, H., Kitamura, K., Itoh, T., and Inoue, R. (1998) Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol. Rev. 78, 811–920.Google Scholar
  15. 15.
    Berridge, M. J., Bootman, M. D., and Lipp, P. (1998) Calcium—a life and death signal. Nature 395, 645–648.CrossRefGoogle Scholar
  16. 16.
    Plank, D. M. and Sussman, M. A. (2005) Impaired intracellular Ca2+ dynamics in live cardiomyocytes revealed by rapid line scan confocal microscopy. Microsc. Microanal. 11, 235–243.CrossRefGoogle Scholar
  17. 17.
    Moreno, D. H. (1999) Molecular and functional diversity of voltage-gated calcium channels. Ann. NY Acad. Sci. 868, 102–117.CrossRefGoogle Scholar
  18. 18.
    Minta, A., Kao, J. P., and Tsien, R. Y. (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178.Google Scholar
  19. 19.
    Haugland, R. P., Spence, M. T. Z., and Johnson, I., eds. (2005) The Handbook: A Guide to Fluorescent Probes and Labeling Technologies. Printed by Invitrogen, CA.Google Scholar
  20. 20.
    Miyawaki, A. (2004) Fluorescent proteins in a new light. Nat. Biotechnol. 11, 1374–1376.CrossRefGoogle Scholar
  21. 21.
    Miyawaki, A. (2005) Innovations in the imaging of brain functions using fluorescent proteins. Neuron 48, 189–199.CrossRefGoogle Scholar
  22. 22.
    Romoser, V. A., Hinkle, P. M., and Persechini, A. (1997) Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13,270–13,274.CrossRefGoogle Scholar
  23. 23.
    Miyawaki, A., Llopis, J., Heim, R., et al. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.CrossRefGoogle Scholar
  24. 24.
    Gao, W., Xing, B., Tsien, R. Y., and Rao, J. (2003) Novel fluorogenic substrates for imaging beta-lactamase gene expression. J. Am. Chem. Soc. 125, 11,146, 11,147.CrossRefGoogle Scholar
  25. 25.
    Zlokarnik, G., Negulescu, P. A., Knapp, T. E., et al. (1998) Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter. Science 279, 84–88.CrossRefGoogle Scholar
  26. 26.
    Johannessen, M., Delghandi, M. P., and Moens, U. (2004) What turns CREB on? Cell Signal 16, 1211–1127.CrossRefGoogle Scholar
  27. 27.
    Drews, J. (2000) Drug discovery: a historical perspective. Science 287, 1960–1964.CrossRefGoogle Scholar
  28. 28.
    England, P. J. (1999) Discovering ion-channel modulators—making the electro-physiologist’s life more interesting. DDT 4, 391–392.Google Scholar
  29. 29.
    Velicelebi, G., Stauderman, K. A., Varney, M. A., Akong, M., Hess, S. D., and Johnson, E. C. (1999) Fluorescence techniques for measuring ion channel activity. Methods Enzmol. 294, 20–47.CrossRefGoogle Scholar
  30. 30.
    Gonzalez, J. E., Oades, K., Leychkis, Y., Harootunian, A., and Negulescu, P. A. (1999) Cell-based assays and instrumentation for screening ion-channel targets. Drug Disc. Today 4, 431–439.CrossRefGoogle Scholar
  31. 31.
    Denyer, J., Worley, J., Cox, B., Allenby, G., and Banks, M. (1998) HTS approaches to voltage-gated ion channel drug discovery. DDT. 7, 323–332.Google Scholar
  32. 32.
    Wuskell, J. P., Boudreau, D., Wei, M. D., et al. (2006) Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges. J. Neurosci. Methods 151, 200–215.CrossRefGoogle Scholar
  33. 33.
    Gonzalez, J. E. and Tsien, R. Y. (1995) Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys. J. 69, 1272–1280.CrossRefGoogle Scholar
  34. 34.
    Gonzalez, J. E. and Tsien, R. Y. (1997) Improved indicators of cell membrane potential that use fluorescence resonance energy transfer. Chem. Biol. 4, 269–277.CrossRefGoogle Scholar
  35. 35.
    González Jesús, E. and Maher, M. P. (2002) Cellular fluorescent indicators and voltage/ion probe reader (VIPR™): tools for ion channel and receptor drug discovery. Receptors Channels 8, 283–295.Google Scholar
  36. 36.
    Epps, D. E., Wolfe, M. L., and Groppi, V. (1994) Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-[(1,3-dibutylbarbituric acid) trimethine oxonol Dibac4(3)] in model systems and cells. Chem. Phys. Lipids 69, 137–150.CrossRefGoogle Scholar
  37. 37.
    Schroeder, K. S. and Neagle, B. D. (1996) FLIPR: a new instrument for accurate, high-throughput optical screening. J. Biomol. Screen. 1, 75–80.CrossRefGoogle Scholar
  38. 38.
    Sullivan, E., Tucker, E. M., and Dale, I. L. (1999) Measurement of [Ca2+] using the fluorometric imaging plate reader (FLIPR). Methods Mol. Biol. 114, 125–133.Google Scholar
  39. 39.
    Bell, T. W. and Hext, N. M. (2004) Supramolecular optical chemosensors for organic analytes. Chem. Soc. Rev. 33, 589–598.Google Scholar
  40. 40.
    Green, D. R. (2005) Apoptotic pathways: ten minutes to dead. Cell. 121, 671–674.CrossRefGoogle Scholar
  41. 41.
    Spierings, D., McStay, G., Saleh, M., et al. (2005) Connected to death: the (unexpurgated) mitochondrial pathway of apoptosis. Science 310, 66, 67.CrossRefGoogle Scholar
  42. 42.
    Watanabe, M., Hitomi, M., van der Wee, K., et al. (2002) The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs. Microsc. Microanal. 8, 375–391.CrossRefGoogle Scholar
  43. 43.
    Gasparri, F., Mariani, M., Sola, F., and Galvani, A. (2004) Quantification of the proliferation indes of human dermal fibroblast cultures with the ArrayScan™ high content screening reader. J. Biomol. Screen. 9, 232–243.CrossRefGoogle Scholar
  44. 44.
    Petronilli, V., Miotto, G., Canton, M., et al. (1999) Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys. J. 76, 725–734.CrossRefGoogle Scholar
  45. 45.
    Smith, P. J., Blunt, N., Wiltshire, M., et al. (2000) Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy. Cytometry. 40, 280–291.CrossRefGoogle Scholar
  46. 46.
    Hansen, J. M., Go, Y. M., and Jones, D. P. (2006) Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling. Annu. Rev. Pharmacol. Toxicol. 46, 215–234.CrossRefGoogle Scholar
  47. 47.
    Granas, C., Lundholt, B. K., Heydorn, A., et al. (2005) High content screening for G protein-coupled receptors using cell-based protein translocation assays. Comb. Chem. High-Throughput Screen. 8, 301–309.CrossRefGoogle Scholar
  48. 48.
    Zucker, R. M. and Price, O. T. (1999) Practical confocal microscopy and the evaluation of system performance. Methods 18, 447–458.CrossRefGoogle Scholar
  49. 49.
    Zucker, R. M. and Price, O. (2001) Evaluation of confocal microscopy system performance. Cytometry. 44, 273–294.CrossRefGoogle Scholar

Copyright information

© Humana Press, Inc. 2007

Authors and Affiliations

  • Michael J. Ignatius
    • 1
  • Jeffrey T. Hung
    • 1
  1. 1.Molecular Probes/InvitrogenEugene

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