In Vitro

, Volume 17, Issue 12, pp 1100–1110 | Cite as

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

  • Hugo O. Jauregui
  • Nancy T. Hayner
  • James L. Driscoll
  • Rhonda Williams-Holland
  • Milton H. Lipsky
  • Pierre M. Galletti
Conference-American Type Culture Collection

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.

Key words

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

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Copyright information

© Tissue Culture Association, Inc 1981

Authors and Affiliations

  • Hugo O. Jauregui
    • 1
  • Nancy T. Hayner
    • 2
  • James L. Driscoll
    • 1
  • Rhonda Williams-Holland
    • 1
  • Milton H. Lipsky
    • 1
  • Pierre M. Galletti
    • 2
  1. 1.Department of PathologyRhode Island HospitalProvidence
  2. 2.Artificial Organ Laboratory, Division of Biology and MedicineBrown UniversityProvidence

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