Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation.

Abstract

The positively charged rhodamine analog rhodamine 123 accumulates specifically in the mitochondria of living cells. In the present work, the uptake of rhodamine 123 by individual lymphocytes undergoing blastogenic transformation in cultures stimulated by phytohemagglutinin was measured by flow cytometry. A severalfold increase in cell ability to accumulate rhodamine 123 was observed during lymphocyte stimulation. Maximal dye uptake, seen on the third day ofcell stimulation, coincided in time with the peak of DNA synthesis (maximal number of cells in the S phase) and mitotic activity. A large intercellular variation among stimulated lymphocytes, with some cells having fluorescence increased as much as 15 times in comparison with nonstimulated lymphocytes, was observed. Whereas the increased uptake ofrhodamine 123 also correlated with the increase in cellular RNA content, the correlation between the dye uptake and cell size (measured by light scatter) was less apparent. As observed by UV microscopy, the increased dye uptake during the blastogenesis was due, to a large extent, to an increase in number of mitochondria per cell. However, an additional increase in rhodamine 123 binding per mitochondrion or per unit of mitochondrial membrane in stimulated cells could not be excluded. The present data indicate that rhodamine 123 may be used as a supravital mitochondrial probe, discriminating between cycling and quiescent cells and having application in sorting functionally distinct cell subpopulations.Johnson et al. (1) have recently reported that the fluorescent laser dye rhodamine 123 directly and selectively stains mitochondria of living cells. These authors observed that the dye is taken up by mitochondria in a variety ofcell types without being accumulated, even transiently, in other cell organelles (i.e., lysosomes, endocytic vesicles). Changes in mitochondrial organization, distribution, and shape induced by viral transformation or colchicine treatment could be visualized easily after staining with rhodamine 123 (1). The dye, reported not to be cytotoxic at concentrations up to 10 ,g/ml, thus may be used supravitally as a mitochondrial probe.We report here that the fluorescence of individual cells stained with rhodamine 123 can be measured by flow cytometry, and we compare the rhodamine 123 binding of noncycling, quiescent lymphocytes with lymphocytes stimulated to proliferate by phytohemagglutinin (PHA). In particular, the uptake of rhodamine 123 is measured and correlated with progression of stimulated lymphocytes through the cell cycle, as analyzed by multiparameter flow cytometry based on simultaneous quantitation of cellular RNA and DNA (2).MATERIALS AND METHODS Cells. Blood was collected by venipuncture of healthy volunteers and the leukocytes were separated by gradient centrifugation on Ficoll/Isopaque (Lymphoprep; Nyegaardo, Oslo, Norway). The mononuclear cells were suspended in Eagle's basal medium containing 15% fetal bovine serum and subcultured in plastic dishes to remove most ofthe monocyte...

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