Mitochondrial assays fluorescent dyes

Function of mitochondria is also commonly monitored as an indicator of cellular toxicity. Mitochondrial uptake and retention of the fluorescent dye, rhodamine 123, can be visualized microscopically. Biochemical measurements of mitochondrial function include the ATP-ADP ratio and dehydrogenase activity with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which yields a colored formazan product upon reduction. The dye, neutral red (3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride), targets lysosomes, and its retention is inversely related to cytotoxicity. Commercially available versions of the MTT and neutral red assays have been adapted to microtiter plate formats to provide highly efficient screening assays. Examples of how cell-type-specific functions can be followed as indicators of cell toxicity are included in Table 8.1. 

Jacohson, J. Duchen, M. R. Heales, S. J. R. Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential imphcations for assays of cardioliponyl acridine orangein and mitochondrial mass. J. Neurochem. 2002, 82, 224—233. 

An important improvement of these assays is the use of fluorescent Pgp substrates with a much higher sensitivity than the anthracyclines. Many of these are dyes that had other applications to monitor cellular functions, such as viability, mitochondrial potential, or pH. We and others have compared extensively many of these dyes for their sensitivity and specificity to detect Pgp function in tumor cell lines (8,11), hematopoietic progenitor cells (12) and AMLs (13). We have chosen to use rhodamine 123 as dye and have validated its use by direct comparison with radiolabeled daunorubicin and vincristine accumulation in AMLs (14). 

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