Mitochondrial assays fluorescence

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. 

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). 

From the aforementioned description of mitochondrial physiology and the MPT, it is clear that pore opening and dissipation of the proton gradient will result in relative alkalinization of the inner mitochondrial space and acidification of the cytoplasm. Fluorescence-based assays to detect altered cellular pH and their use in apoptosis will be addressed below, as will alterations in intracellular calcium concentrations. Similiarly, although the mitochondrial electron-transport chain is an important source of reactive oxygen... 

Fig. 2. Cytogram of a combined assay for NAD(P)H levels (UV-excited blue autofluorescence) and mitochondrial membrane potential (CMXRosamine/ MitoTracker Green FM fluorescence). Signals representing normal and compromomised cells are labeled.

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

Woollacott, A. J. Simpson, R. B. High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells. J. Biomol. Screen. 2001, 6, 413 20. 

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. 

The cytotoxicities of both DNP and SDS are presented in Figure 6A and B. For each compound, one can determine the cytotoxic concentration exerting 50% cell damage, i.e. 50% decrease of the fluorescent signal of control cells. Depending on the target, membrane or mitochondrial damage, the best test for SDS is the calcein-AM assay, whereas the Alamar blue assay is the most sensitive one for DNP. 

Blattner, J. R. He, L. Lemasters, J. J. Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader. Anal. Biochem. 2001,295, 220-226. 

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