6th ESACP Congress, Heidelberg, April 7-11, 1999

A031
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

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.