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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A031
Cell-mediated reduction of tetrazolium salts, including MTT, XTT, MTS, NBT,
NTV, INT, in the presence and absence of intermediate electron carriers is
used as a convenient test for animal or bacterial cell viability.
Bioreduction of tetrazolium is considered an alternative to a clonogenic
assay and a thymidine incorporation assay. However, correlation between
clonogenic potential and capacity to reduce tetrazolium has not been
demonstrated convincingly. Moreover, despite a wide use of tetrazolium
viability assays, the mechanism and subcellular localization of reducing
systems or species in viable intact cells have not been fully elucidated.
We report evidence demonstrating that a tetrazolium salt CTC
(5-cyano-2,3,-ditolyl tetrazolium chloride) can be reduced to a corresponding
formazan in the presence as weil as in the absence of an electron carrier
(Meldola's Blue) by viable HepG2 human hepatoma cells. Detection of formazan
was achieved by confocal microscopy. CTC-formazan was deposited in the
regions of plasma membranes, probably at the outer surface. Our data indicate
that electron donors active in reduction of CTC in the presence of an
electron carrier may be located in the intracellular compartment as well as
in plasma membranes. In the absence of an electron carrier, however, the
reduction may be performed exclusively by plasma membrane-associated
oxidoreductases.
REDUCTION OF A TETRAZOLIUM SALT, CTC, BY INTACT HEPG2 HUMAN HEPATOMA
CELLS - SUBCELLULAR LOCALIZATION OF REDUCING SYSTEMS
Bernas T, Dobrucki J
Laboratory of Confocal Microscopy, Department of Biophysics,
Institute of Molecular Biology, Jagiellonian University, Krakow, Poland