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Solid Tissue Dispersal for Cytokinetic Analyses

  • Maria G. Pallavicini
Protocol
Part of the Biological Methods book series (BM)

Abstract

The dispersal of solid tissues into a single-cell suspension has become an integral component of many current techniques for quantitative cytokinetic analysis of both normal and neoplastic tissues. This is particularly evident in the application of flow cytometric (FCM) procedures for the quantitative analysis of DNA content (see ref. 36), measurements of intracellular components [i.e., bromodeoxyuridine incorporated into DNA (15)], and identification and purification of subpopulations for subsequent cytokinetic studies (42,71). In addition most assay systems for quantitation of the fraction of clonogenic cells, as well as many biochemical/molecular analyses (i.e., incorporation of tritiated thymidine, [3H]-TdR, into DNA), are done on a per cell basis, and thus utilize cells in suspension.

Keywords

Neutral Protease Solid Tissue Tumor Cell Suspension Tissue Dispersal Dispersal Protocol 
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.

References

  1. 1.
    Barlogie, B., Gohde, W., Johnston, A., Smallwood, L., Schuman, J., Drewinko, B., and Freireich, D. J. Determination of ploidy and proliferative characteristics of human solid tumors by pulse cytophotometry. Cancer Res., 38: 3333–3339, 1978.PubMedGoogle Scholar
  2. 2.
    Bashor, M. M. Dispersion and disruption of tissues. In: (W. B. Jakoby and I. H. Pasten, eds.), Methods in Enzymology, vol. LVIII, New York: Academic, 1979.Google Scholar
  3. 3.
    Bauer, F. W., Boezeman, J., and deGrood, R. M. Cell cycle analysis in normal and pathologic skin. Pulse-Cytophotometry, III: 533–538, 1978.Google Scholar
  4. 4.
    Berry, M. N. and Friend, D. S. High-yield preparation of isolated rat liver parenchymal cells. J. Cell Biol., 43: 506–520, 1969.PubMedCrossRefGoogle Scholar
  5. 5.
    Berwick, L. and Corman, D. R. Some chemical factors in cellular adhesion and stickiness. Cancer Res., 22: 982–986, 1962.Google Scholar
  6. 6.
    Boyse, E. A. A method for the production of viable cell suspensions from solid tumors. Transplant. Bull., 7: 100–104, 1960.PubMedGoogle Scholar
  7. 7.
    Brown, J. M., Twentyman, P. R., and Zamvil, S. S. Response of the RIF-1 tumor in vitro and in C3H/Km mice to x-irradiation (cell survival, regrowth delay, and tumor control), chemotherapeutic agents, and activated macrophages. J. Natl. Cancer Inst., 64: 605–611, 1980.PubMedGoogle Scholar
  8. 8.
    Burlington, H. and Cronkite, E. P. Characteristics of cell cultures derived from renal glomeruli. Proc. Soc. Exp. Biol. Med., 142: 143–149, 1973.PubMedGoogle Scholar
  9. 9.
    Burns, E. R., Bagwell, B. C, Hinson, W. G., Pipkin, J. L., and Hudson, J. L. Preparation stability of sixteen murine tissues organs for flow cytometric cell cycle analysis. Cytometry, 4: 150–160, 1983.PubMedCrossRefGoogle Scholar
  10. 10.
    Camazine, S. M., Ryan, G. B., Unanue, E. R., and Karnovsky, N. J. Isolation of phagocytic cells from the rat renal glomerulus. Lab. Invest., 35: 315–326, 1976.PubMedGoogle Scholar
  11. 11.
    Cook, G. M. W., Heard, D. H., and Seamna, G. V. F. A sialomuco-peptide liberated by trypsin from the human erythrocyte. Nature, 138: 1011–1012, 1960.CrossRefGoogle Scholar
  12. 12.
    Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M. R. Lymphocyte stimulation: A rapid multiparameter analysis. Proc. Natl. Acad. Sci. USA, 73: 2881–2884, 1976.PubMedCrossRefGoogle Scholar
  13. 13.
    Darzynkiewicz, Z., Traganos, F., and Melamed, M. R. Detergent treatment as an alternative to cell fixation for flow cytometry. J. Histochem. Cytochem., 29: 329–330, 1981.PubMedGoogle Scholar
  14. 14.
    Dethlefsen, L. A., Gray, J. W., George, Y. S., and Johnson, S. Flow cytometric analysis of the perturbed cellular kinetics of solid-tumors: Problems and promises. In: (W. Gohde, J. Schumann, and Th. Bucher, eds.), Pulse-Cytophotometry, Ghent, Belgium: European Press, 1976.Google Scholar
  15. 15.
    Dolbeare, F., Gratzner, H., Pallavicini, M. G., and Gray, J. W. Flow cytometric analysis of total DNA content incorporated bromodeoxyuridine. Proc. Natl. Acad. Sci. USA, 80: 5573–5577, 1983.PubMedCrossRefGoogle Scholar
  16. 16.
    Finkelstein, J. N. and Shapiro, D. L. Isolation of Type II alveolar epithelial cells using low protease concentrations. Lung, 160: 85–98, 1982.PubMedCrossRefGoogle Scholar
  17. 17.
    Goodenough, D. A. and Gilula, N. B. The splitting of hepatocyte gap junctions and zonae occludentes with hypertonic disaccharide. J. Cell Biol., 61: 575–590, 1974.PubMedCrossRefGoogle Scholar
  18. 18.
    Gottschalk, A., Belyavin, G., and Biddle, F. In: (A. Gottschalk, ed.), Glycoproteins: Their Composition, Structure, and Function, 2nd Ed., part B., Amsterdam: Elsevier, 1972.Google Scholar
  19. 19.
    Hamburger, A. W., White, C. P., and Tencer, K. Effect of enzymatic disaggregation on proliferation of human tumor cells in soft agar. J. Natl. Cancer Inst., 68: 945–949, 1982.PubMedGoogle Scholar
  20. 20.
    Harris, C. C. and Leone, C. A. Some effects of EDTA and tetraphenylboron on the ultrastructure of mitochondria in mouse liver cells. J. Cell Biol., 28: 405–408, 1966.PubMedCrossRefGoogle Scholar
  21. 21.
    Harrison, D. D. and Webster, H. L. The preparation of isolated crypt cells. Exp. Cell Res., 55: 257–260, 1969.PubMedCrossRefGoogle Scholar
  22. 22.
    Hill, R. P. and Pallavicini, M. G. Hypoxia and the radiation response of tumors. In: (H. I. Bicher and D. F. Bruley, eds.), Oxygen Transport to Tissue, vol. IV, New York: Plenum, 1983.Google Scholar
  23. 23.
    Hommes, F. A., Draisman, M. I., and Molenaar, I. Preparation and some properties of isolated rat liver cells. Biochem. Biophys. Acta., 222: 361–371, 1970.PubMedGoogle Scholar
  24. 24.
    Hosick, H. L. and Strohman, R. Changes in ribosome-polyribosome balance in chick muscle cells during tissue dissociation, development in culture and exposure to simplified culture medium. J. Cell Physiol., 77: 145–156, 1971.PubMedCrossRefGoogle Scholar
  25. 25.
    Kikkawa, Y., Yoneda, K., Smith, F., Packard, B., and Suzuki, K. The type II epithelial cells of the lung. II. Chemical composition and phospholipid synthesis. Lab. Invest., 32: 295–302, 1975.PubMedGoogle Scholar
  26. 26.
    Kleinman, H. K., Klebe, R. J., and Martin, G. R. Role of collagenous matrices in the adhesion and growth of cells. J. Cell Biol., 88: 473–485, 1981.PubMedCrossRefGoogle Scholar
  27. 27.
    Kreisberg, J.I. Isolation and culture of homogeneous populations of glomerular cell types. In: (T. G. Pretlow and T. P. Pretlaw, eds.), Cell Separation. Methods and Selected Applications, vol. I, New York: Academic, 1982.Google Scholar
  28. 28.
    Laerum, O. D. Oxygen consumption of basal and differentiating cells from hairless mouse epidermis. J. Invest. Dermatol., 52: 204–211, 1969.PubMedGoogle Scholar
  29. 29.
    Lawson, A. J., Smit, R. A., Jjefers, N. A., and Osborne, J. W. Isolation of rat intestinal crypt cells. Cell Tissue Kinet., 15: 69–80, 1982.PubMedGoogle Scholar
  30. 30.
    Levinson, C. and Green, J. W. Cellular injury resulting from tissue disaggregation. Exp. Cell Res., 39: 309–317, 1965.PubMedCrossRefGoogle Scholar
  31. 31.
    Lewin, M. J. M., Cheret, A. M., and Sachs, G. Separation of individual cells from the fundic gastric mucosa. In: (T. G. Pretlow and T. P. Pretlow, eds.), Cell Separation: Methods and Selected Applications, vol. I, New York: Academic, 1982.Google Scholar
  32. 32.
    Madden, R. E. and Burk, D. Production of viable cell suspensions from solid tumors. J. Natl. Cancer Inst., 27: 841–861, 1961.PubMedGoogle Scholar
  33. 33.
    Magee, W. E., Sheek, N. R., and Sagik, B. P. Methods of harvesting mammalian cells grown in tissue culture. Proc. Soc. Exp. Biol. Med., 99: 390–392, 1958.PubMedGoogle Scholar
  34. 34.
    McDivitt, R. W., Stone, K. R., and Meyer, J. S. A method for dissociation of viable human breast cancer cells that produces flow cytometric kinetic information similar to that obtained by thymidine labeling. Cancer Res., 44: 2628–2633, 1984.PubMedGoogle Scholar
  35. 35.
    Mead, J. S., Horan, P. K., and Wheeless, L. L. Syringing as a method of tissue dispersal. I. Effect on intermediate and superficial squamous cells. Acta Cytol., 22: 86–90, 1978.PubMedGoogle Scholar
  36. 36.
    Melamed, M. R., Mullaney, P. F., and Mendelsohn, M. L., eds. Flow Cytometry and Sorting. New York: John Wiley, 1979.Google Scholar
  37. 37.
    Milas, L. and Mujagic, H. The effect of spleenectomy on fibrosarcoma “metastasis” in lungs of mice. Int. J. Cancer, 11: 186–190, 1973.PubMedCrossRefGoogle Scholar
  38. 38.
    Moran, J. and Cohen, L. Use of papain in the preparation of adult skeletal muscle for tissue culture. In Vitro, 10: 188–190, 1974.CrossRefGoogle Scholar
  39. 39.
    Pallavicini, M. G., Cohen, A. M., Dethlefsen, L. A., and Gray, J. W. Dispersal of solid tumors for flow cytometric (FCM) analysis. In: (D. Lutz, ed.), Pulse-Cytophotometry, part III, Ghent, Belgium: European Press, 1978.Google Scholar
  40. 40.
    Pallavicini, M. G., Folstad, L. J., and Dunbar, C. Solid KHT tumor dispersal for flow cytometric cell kinetic analysis. Cytometry, 2: 54–58, 1981.PubMedCrossRefGoogle Scholar
  41. 41.
    Pallavicini, M. G., Gray, J. W., and Folstad, L. J. Quantitative analysis of the cytokinetic response of KHT tumors in vivo to 1-β-D-arabino-furanosylcytosine. Cancer Res., 42: 3125–3131, 1982.PubMedGoogle Scholar
  42. 42.
    Pallavicini, M. G., Ng, C. R., and Gray, J. W. Bivariate flow cytometric analysis of murine intestinal epithelial cells for cytokinetic studies. Cytometry, 5: 55–62, 1984.PubMedCrossRefGoogle Scholar
  43. 43.
    Penning, J. J. and Levan, J. H. A modified enzymatic technique for production of cell suspensions of a murine fibrosarcoma. J. Natl. Cancer Inst., 66: 85–87, 1981.PubMedGoogle Scholar
  44. 44.
    Phillips, H. J. Some metabolic changes resulting from treating kidney tissue with trypsin. Can. J. Biochem., 48: 1495–1504, 1967.Google Scholar
  45. 45.
    Piwnicka, M., Darzynkiewicz, Z., and Melamed, M. R. RNA and DNA content of isolated nuclei measured by multiparameter flow cytometry. Cytometry, 3: 269–275, 1983.PubMedCrossRefGoogle Scholar
  46. 46.
    Pollack, A., Prudhomme, D. C, Greenstein, D. B., Irwin, G. C, Claffin, A. J., and Block, N. C. Flow cytometric analysis of RNA content in different cell populations using pyronin y methyl green. Cytometry, 3: 28–35, 1982.PubMedCrossRefGoogle Scholar
  47. 47.
    Rappaport, C. and Howze, G. Dissociation of adult mouse liver by sodium tetraphenylboron, a potassium complexing agent. Proc. Soc. Exp. Biol. Med., 121: 1010–1016, 1966.PubMedGoogle Scholar
  48. 48.
    Rasey, J. S. and Nelson, N. J. Effect of tumor disaggregation on results of in vitro cell survival assay after in vivo treatment of the EMT6 tumor: X-rays, cyclophosphamide, and bleomycin. In Vitro, 16: 547–553, 1980.PubMedCrossRefGoogle Scholar
  49. 49.
    Reimann, J., Ehman, D., and Miller, R. G. Differential binding of lectins to lymphopoietic myeolopoietic cells in murine marrow as revealed by flow cytometry. Cytometry, 5: 194–203, 1984.PubMedCrossRefGoogle Scholar
  50. 50.
    Reinhold, H. S. A cell dispersal technique for use in quantitative transplantation studies with solid tumors. Eur. J. Cancer, 1: 67–71, 1965.PubMedGoogle Scholar
  51. 51.
    Rinaldini, L. The isolation of living cells from animal tissues. Int. Rev. Cytol., 7: 587–647, 1958.CrossRefGoogle Scholar
  52. 52.
    Rockwell, S., Kallman, R. F., and Fajando, L. F. Characteristics of a serially transplanted mouse mammary tumor and its tissue-culture adopted derivatives. J. Natl. Cancer Inst., 49: 735–749, 1972.PubMedGoogle Scholar
  53. 53.
    Rockwell, S. and Kallman, R. Growth and cell population kinetics of single and multiple KHT sarcomas. Cell Tissue Kinet., 5: 449–457, 1972.PubMedGoogle Scholar
  54. 54.
    Roters, M., Linden, W. A., and Heienbrok, W. Comparison of three different methods for the preparation of human tumors for flow cytometry (FCM). In: (D. Lutz, ed.), Pulse Cytometry, part IV, Ghent, Belgium: European Press, 1978.Google Scholar
  55. 55.
    Russell, S. W., Doe, W. F., Hoskins, R. G., and Cochrane, C. G. Inflammatory cells in solid murine neoplasms. I. Tumor disaggregation and identification of constituent inflammatory cells. Int. J. Cancer, 18: 322–330, 1976.PubMedCrossRefGoogle Scholar
  56. 56.
    Schapp, G. H.,Josselin de Jong, J. E., and Jonkind, J. F. Fluorescence polarization of six membrane probes in embryonal carcinoma cells after differentiation as measured on a FACS II cell sorter. Cytometry, 5: 188–193, 1984.CrossRefGoogle Scholar
  57. 57.
    Seglen, P. O. Preparation of rat liver cells. Exp. Cell Res., 74: 377–389, 1971.Google Scholar
  58. 58.
    Severin, E., Zold, H., and Spies, I. Proposals for isolation and further handling of liver cells in flow-cytometry. In: (D. Lutz, ed.), Pulse-Cytometry, part III, Belgium: Ghent, European Press, 1978.Google Scholar
  59. 59.
    Slocum, H. K., Pavelic, Z. P., Kanter, P. M., Nowak, N. J., and Rustum, Y. M. The soft agar clonogenicity and characterization of cells obtained from human solid tumors by mechanical and enzymatic means. Cancer Chemother. Pharmacol., 6: 219–225, 1981.PubMedCrossRefGoogle Scholar
  60. 60.
    Slocum, H. K., Pavelic, Z. P., Rustum, Y. M., Creaven, P. J., Karskousis, C., Takita, H., and Greco, W. R. Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma, and lung tumors. Cancer Res., 41: 1428–1434, 1981.PubMedGoogle Scholar
  61. 61.
    Snow, C. and Allen, A. The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetraacetate treatment of baby hamster cells in tissue culture. Biochem. J., 119: 707–714, 1970.PubMedGoogle Scholar
  62. 62.
    Steinberg, M. S. The role of temperature in the control of aggregation of dissociated embryonic cells. Exp. Cell Res., 21: 1–10, 1961.Google Scholar
  63. 63.
    Thomson, J. E. and Rauth, A. M. An in vitro assay to measure the viability of KHT tumor cells not previously exposed to culture conditions. Radiat. Res., 58: 262–276, 1974.PubMedCrossRefGoogle Scholar
  64. 64.
    Thornwaite, J. T., Sugarbaker, E. V., and Temple, W. J. Preparation of tissues for DNA flow cytometric analysis. Cytometry, 1: 229–237, 1980.CrossRefGoogle Scholar
  65. 65.
    Traganos, F., Darzynkiewicz, Z., Sharpless, T., and Melamed, M. R. Simultaneous staining of ribonucleic and deoxyribonucleic acid in unfixed cells using acridine orange in a flow cytofluorometric system. J. Histochem. Cytochem., 25: 46–56, 1977.PubMedGoogle Scholar
  66. 66.
    Trotman, C. N. A. Isolation of gastrointestinal mucosa. In: (E. Reid, ed.), Cell Populations, Chichester, England: Horwood/Wiley, 1979.Google Scholar
  67. 67.
    Twentyman, P. R. An artefact in clonogenic assays of bleomycin cytotoxicity. Br. J. Cancer, 36: 642–644, 1977.PubMedCrossRefGoogle Scholar
  68. 68.
    Twentyman, R. R. and Yuhas, J. M. Use of bacterial neutral protease for disaggregation of mouse tumors and multicellular tumor spheroids. Cancer Lett., 9: 225–228, 1980.PubMedCrossRefGoogle Scholar
  69. 69.
    Vindelov, L. L. Flow microflurometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. Virchows Arch. Cell Pathol., 24: 227–242, 1977.Google Scholar
  70. 70.
    Vindelov, L. L., Christensen, I. J., and Nissen, N. I. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry, 3: 323–327, 1983.PubMedCrossRefGoogle Scholar
  71. 71.
    Visser, J. W. M. and Bol, S. J. L. A two-step procedure for obtaining 80-fold enriched suspensions of murine pluripotent hemopoietic stem cells. Stem Cells, 1: 240–249, 1981.Google Scholar
  72. 72.
    Wake, N., Slocum, H. R., Rustum, Y. M., Matsui, S., and Sandberg, A. A. Chromosomes and causation of human cancer and leukemia XLIV. A method for chromosome analysis of tumors. Cancer Gen. Cytogen., 3: 1–10, 1981.CrossRefGoogle Scholar
  73. 73.
    Waymouth, C. To disaggregate or not to disaggregate: Injury and cell disaggregation, transient or permanent? In Vitro, 39: 97–111, 1974.CrossRefGoogle Scholar
  74. 74.
    Waymouth, C. Obtaining cell suspensions from animal tissues. In: (T. G. Pretlow and T. P. Pretlow, eds.), Cell Separation: Methods and Selected Applications, vol I., New York: Academic, 1982.Google Scholar
  75. 75.
    Weiser, M. Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J. Biol. Chem., 248: 2536–2541, 1973.PubMedGoogle Scholar
  76. 76.
    Weiser, R. S., Heise, E., Mclvor, K., Han, S., and Granger, G. In vitro activities of immune macrophages. In: (R. T. Smith and R. A. Good, eds.), Cellular Recognition, New York: Appleton-Century-Crofts, 1969.Google Scholar

Copyright information

© The Humana Press Inc. 1987

Authors and Affiliations

  • Maria G. Pallavicini
    • 1
  1. 1.Biomedical Sciences Division, Lawrence Livermore National LaboratoryUniversity of CaliforniaLivermore

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