Journal of tissue culture methods

, Volume 13, Issue 4, pp 257–259 | Cite as

An efficient and simple method of non-enzymatic detachment of monolayer cultures into single cell suspension

  • K. H. Sit
  • K. P. Wong
  • B. H. Bay
Contributed Papers

Summary

Activating the Na+/H+ antiporter causes a distinctive and rapid endocytosis which internalizes the plasma membrane leading to cell retraction and detachment as rounded cells with much reduced surface area. Antiport activation is by a combination of two individually effective motivations, viz. a) steep [Na+] and [H+] gradients across the plasma membrane and b) allosteric activation by second messengers initiated with simple inorganic sulphate. The dissociated cells are as viable as those released by trypsinization. Thus it provides an effective enzyme-free alternative to trypsin digestion in cell dissociation.

Key words

cell dissociation detachment rounding Na+/H+ antiport endocytosis sulphate 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

V. References

  1. 1.
    Anghileri, L. J. Dermietzel. Cell coat in tumor cells, effects of trypsin and EDTA: a biological and morphological study. Oncology 33:17–23; 1976.Google Scholar
  2. 2.
    Chinkers, M.; McKanna, J. A.; Cohen, S. Rapid induction of morphological changes in human carcinoma cells A431 by epidermal growth factor. J. Cell Biol. 83:260–265; 1979.CrossRefPubMedGoogle Scholar
  3. 3.
    Chinkers, M.; McKanna, J. A.; Cohen, S. Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor. J. Cell Biol. 88:422–428; 1981.CrossRefPubMedGoogle Scholar
  4. 4.
    Defize, L. H.; Moolenaar, W. H.; Van der Saag, P. T., et al. Dissociation of cellular responses to epidermal growth factor using anti-receptor monoclonal antibodies. EMBO J. 5:1187–1192; 1986.PubMedGoogle Scholar
  5. 5.
    Moolenaar, W. H. Ionic signals in the action of growth. Ann. NY Acad. Sci. 551:321–333; 1989.Google Scholar
  6. 6.
    Pharmacia Fine Chemicals. Microcarrier cell culture, principles & methods. Uppsala, Sweden: Pharmacia Fine Chemicals AB; 1981.Google Scholar
  7. 7.
    Ruoslahti, E. Fibronectin and its receptors. Ann. Rev. Biochem. 57:375–413; 1988.PubMedGoogle Scholar
  8. 8.
    Ruoslahti, E. Proteoglycans in cell regulation. J. Biol. Chem. 264:13369–133372; 1989.PubMedGoogle Scholar
  9. 9.
    Ruoslahti, E.; Pierschbacher, M. D. New perspectives in cell adhesion: RGD and integrins. Science 238:491–497; 1987.PubMedGoogle Scholar
  10. 10.
    Ryan, U. S.; Mortara, M.; Whitaker, C. Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes. Tissue Cell 12:619–635; 1980.CrossRefPubMedGoogle Scholar
  11. 11.
    Ryan, U. S.; Maxwell, G. Isolation, culture and subculture of bovine pulmonary artery endothelial cells: mechanical methods. J Tissue Cult. Methods 10:3–5; 1986.Google Scholar
  12. 12.
    Schlessinger, J.; Gieger, B. Epidermal growth factor induces redistribution of actin andα-actinin in human epidermal carcinoma cells. Exp Cell Res. 134:273–279; 1981.CrossRefPubMedGoogle Scholar
  13. 13.
    Sit, K. H.; Wong, K. P. Induced interphase cell retraction: its reversal and EGF potentiation. Tissue Cell 21:321–333; 1989.CrossRefPubMedGoogle Scholar
  14. 14.
    Sit, K. H.; Wong, K. P. Sulphate induces very fast cell rounding and detachment. Biochim. Biophys. Acta. 1092:180–183; 1991.PubMedGoogle Scholar
  15. 15.
    Sit, K. H.; Bay, B. H.; Wong, K. P. Antiport mediated cell retraction: viable rounding and distinctive endocytosis. Tissue Cell 22:785–802; 1990.CrossRefPubMedGoogle Scholar
  16. 16.
    Snow, C.; Allan, A. The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediametetra-acetate treatment of baby hamster cells in culture. Biochem. J. 119:707–714; 1970.PubMedGoogle Scholar

Copyright information

© Tissue Culture Association 1991

Authors and Affiliations

  • K. H. Sit
    • 1
  • K. P. Wong
    • 1
  • B. H. Bay
    • 1
  1. 1.Department of Anatomy, Faculty of MedicineNational University of SingaporeKent RidgeSingapore

Personalised recommendations