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J Microsc. 1991 Aug;163(Pt 2):233-41.

Accumulation of fluorescent non-cationic probes in mitochondria of cultured cells: observations, a proposed mechanism, and some implications.

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Department of Biomedical Science, The University, Sheffield, U.K.


Cultured rat fibroblasts were exposed to millimolar concentrations of forty-four non-cationic fluorescent probes, of very varied physico-chemical properties. Mitochondrial staining occurred with nineteen of these probes, nine of which were nominally anionic and ten nominally non-ionic. All nineteen were in fact lipophilic weak acids. Using structural parameters these could be specified numerically as follows: electric charge less than or equal to 0; log P (less-ionized form) less than 0; and pKa approximately 7. In addition to these structural variables, dye concentration and the time of exposure of cells to probes were significant factors for the staining of mitochondria. Accumulation of these compounds can be understood in terms of ion-trapping of hydrophilic salts of lipophilic weak acids, due to the internal pH of respiring mitochondria being higher than the cytosolic pH. As a case example of the application of this approach, the mode of action of many inhibitors of mitochondrial anabolism is discussed in terms of the mechanisms introduced here.

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