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Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

In Vitro volume 17, pages 1100–1110 (1981)Cite this article

Summary

Leakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2±1.5×10−6 IU assayed at 37°C for 25 preparations of isolated hepatocytes.

The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells.

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Affiliations

  1. Department of Pathology, Rhode Island Hospital, 02902, Providence, Rhode Island

    Hugo O. Jauregui M.D., Ph.D., James L. Driscoll, Rhonda Williams-Holland & Milton H. Lipsky

  2. Artificial Organ Laboratory, Division of Biology and Medicine, Brown University, 02902, Providence, Rhode Island

    Nancy T. Hayner & Pierre M. Galletti

Authors
  1. Hugo O. Jauregui M.D., Ph.D.
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  2. Nancy T. Hayner
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  3. James L. Driscoll
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  4. Rhonda Williams-Holland
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  5. Milton H. Lipsky
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  6. Pierre M. Galletti
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Additional information

This study was supported in part by Grants HL-11945-11 and 1-RO1-AM 26520-01A1 from the National Institutes of Health, Bethesda, MD.

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Jauregui, H.O., Hayner, N.T., Driscoll, J.L. et al. Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures. In Vitro 17, 1100–1110 (1981). https://doi.org/10.1007/BF02618612

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  • DOI: https://doi.org/10.1007/BF02618612

Key words

  • trypan blue
  • lactate dehydrogenase
  • hepatocyte cultures
  • isolated hepatocytes
  • vital dye staining
  • enzyme leakage