QUALITATIVE MEASUREMENTS OF THE MITOCHONDRIAL-MEMBRANE POTENTIAL INSITU IN EHRLICH ASCITES TUMOR-CELLS USING THE SAFRANINE METHOD

1. 1. A differential spectrum with a decrease in the absorbance at 524 nm appears when Ehrlich ascites tumour cells are added into a medium containing safranine. This spectral change is similar to that which occurs upon polarisation of the membrane of isolated mitochondria. The maximal change occurs within 15-20 min after cell addition. Additions of KCN, rotenone, antimycin A or carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the cell suspension do not reverse the response but result in a spectrum in which a peak appears at higher wavelengths (540 nm). This suggests that upon deenergization of cells safranine is transferred to a milieu of low polarity. 2. 2. Addition of air to anaerobic cells or glucose to cells whose respiration is inhibited by rotenone, also produces a shift in the safranine spectrum typical to that which occurs upon energization of mitochondria. The response of glucose is insensitive to KCN but abolished by dicyclohexylcarbodiimide, suggesting that ATP produced by glycolysis is responsible for this spectral change. 3. 3. If succinate is added to the cell suspension no response of this substrate is seen with intact cells. However, if cells are pretreated with dextran sulfate, which makes the plasma membrane permeable to small molecules a fast shift is produced upon succinate addition. 4. The results suggest that the spectral changes are signals from mitochondria within the cell. Thus by measuring these changes in the safranine spectrum information about the energy metabolism of cells and how mitochondria function in situ can be obtained. © 1979.