INTERACTIONS OF RHODAMINE-123 WITH LIVING CELLS STUDIED BY FLOW-CYTOMETRY

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  • 1 January 1982
    • journal article
    • research article
    • Vol. 42  (3) , 799-806
Abstract
The cationic fluorochrome rhodamine 123 (R123), reported to bind specifically to mitochondria of living cells, was investigated with respect to its uptake by a variety of cell types [Chinese hamster ovary CHO cells, mouse leukemia L1210 cells, Friend murine leukemia virus-transformed cells] in various functional states and the subsequent effect of the dye on cell growth. The emission spectrum of R123 taken up by cells undergoes a 12 nm red shift, suggesting formation of a complex. Cells accumulate R123 rapidly; near maximum binding is reached after 5-10 min, regardless of the temperature (0-37.degree. C) of incubation. There is a dose-dependent relationship between R123 concentration in the medium and the dye accumulation in the cell that covers the range of 0.1-10.0 and 0.1-5.0 .mu.g R123 /ml under equilibrium and nonequilibrium conditions, respectively. Some leakage of the dye from cells occurs following their transfer into dye-free medium. Despite the leakage the intracellular dye can be detected after at least 2 cell divisions, indicating that the R123-labeled cells divide, labeled mitochondria are distributed into the daughter cells during division and R123 may be used as a cell tracer. Cell death often is accompanied by a transient increase in R123 fluorescence. Dead cells exhibit either uniform, strong fluorescence or show a patchy labeling pattern suggesting swollen mitochondria. With time (4-8 h) dead cells lose ability to retain R123 and lyse. R123 uptake by living cells is increased during the transition from quiescence into the cycle, and a decrease is seen when Friend leukemia cells undergo erythroid differentiation; in all cases, changes in R123 uptake are correlated with changes in cellular RNA content. Simultaneous cell staining with R123 and ethidium or propidium provides a rapid assay of the viability of the cells and their metabolic state, i.e., as related to proliferation or motility. Pulse-labeling of cells with .ltoreq. 10 .mu.g R123/ml has no significant effect on their immediate growth and cloning efficiency. In the continuous presence of R123, cells become specifically arrested in the G1A compartment, i.e., in early G1 phase. Detailed analysis of cell cycle kinetics reveals that cell progression through all phases is slowed 4 h after addition of R123. Cell exit from G1A is affected as early as 2 h following addition of R123; with time the cells are unable to leave this compartment at all. Uncharged rhodamine dyes (rhodamine 110 and rhodamine B) do not accumulate in mitochondria and are without effect on the cell cycle. The cytostatic effect of R123 is discussed in light of the dye specificity for mitochondrial membranes and the disruption of cell energy metabolism resulting in the inability of the cells to attain a critical content of essential components (i.e., ribosomal RNA) necessary for cell entrance into the prereplicative (G1B) compartment of G1 phase.