Localization of mitochondria in living cells with rhodamine 123.

Abstract

The laser dye rhodamine 123 is shown to be a specific probe, for the localization of mitochondria in living cells.By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment.Since the classical investigations of the. mitochondrion some 90 years ago, much work has been carried out on the structure and function of this complex organelle. Extensive biochemical studies of mitochondria have proved that they play a cardinal role in the generation of energy essential for the survival and proliferation of eukaryotic cells (1-4). As intracellular organelles, mitochondria show remarkable plasticity, mobility, and morphological heterogeneity (5-13). Mitochondrial morphology is influenced by the metabolic state of the cell, cell cycle, cellular development and differentiation, and by pathological states (14)(15)(16)(17)(18)(19)(20). In addition, both morphological and functional changes in mitochondria have been shown to occur in conjunction with neoplastic transformation (21).Although isolated mitochondria and mitochondria in fixed cells have been extensively investigated, much less attention has been paid to mitochondria in living cells. Previous investigations of mitochondria in living cells have been hampered by the lack of techniques allowing high-resolution visualization of these organelles. Use of Janus Green B, a dye relatively specific for mitochondria, aids somewhat in their recognition but also causes mitochondrial distortion and cytotoxic effects (5,22 sponsible for mitochondrion-specific staining in such a preparation has been characterized as rhodamine 3B (unpublished data). This finding prompted us to screen various rhodamine compounds for mitochondrion-specific staining. The results reported here describe the use of the laser dye rhodamine 123 as a specific fluorescent probe for mitochondria in living cells. Rhodamine 123 stains mitochondria directly (without passage through endocytic vesicles and lysosomes) and provides lowbackground high-resolution fluorescent images of mitochondria without apparent cytotoxic effects. MATERIALS AND METHODSThe purified laser dye rhodamine 123 (Eastman) was dissolved in double-distilled water at a concentration of 1 mg/ml and subsequently diluted to 10 ,g/ml in Dulbecco's modified Eagle's medium (GIBCO). Cultured cells grown on 12-mm round glass coverslips (Rochester Scientific, Rochester, NY) were incubated with rhodamine 123 (10 ,ug/ml) for 30 min in a 10% CO2 incubator at 37°C. Coverslips were then rinsed through three 5-ml changes of medium (5 min per rinse) and mounted in medium supplemented with 10% fetal calf serum (GIBCO) on a live-cell observation chamber prep...

Intracellular binding of fluorescein in lymphocytes

“…However, others (10) have found that mitochondria of living cells are specifically stained by positively charged dyes including rhodamine 123 and rhodamine 6G, whereas uncharged rhodamines and fluorescein, which is negatively charged at physiologic pH, do not bind. Our own microscopic observations of liver cells indicated that FDA generated fluorescein accumulated preferentially in lysosomes in accordance with the high concentrations of hydrolytic enzymes in these organelles.…”

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

“…Rh 123 is a cationic, cell-permeant fluorochrome characterized by rapid cellular uptake and equilibration (within a few minutes), that has been extensively used to stain mitochondria [78]. However, the application of Rh 123 to the measurement of m is limited by the quenching of the dye at high concentrations [77,79].…”

Coincident loss of mitochondria and nuclei during lens fiber cell differentiation

“…Lens slices, supported on coverslips, were stained for 2-3 min with a 10 pgiml solution of rhodamine 123 in 0.15 M NaC1. Rhodamine 123 is a highly specific, relatively non-toxic, vital stain (Johnson et al, 1980) that distributes into the mitochondrial matrix according to the membrane potential (Emaus et al, 1986). Following staining, lens slices were rinsed with saline and examined on the confocal microscope.…”