CHLOROMETHYL-X-ROSAMINE
In a recent issue of Cytometry, the interpretation of flow cytometric data obtained after chloromethyl-X-rosamine (CMXRos) staining of apoptotic cells became the subject of a debate (1,3). Ferlini et al. (1) analyzed data in an article by Macho et al. in Cytometry (2) and found discrepant changes in ''red'' fluorescence in experiments in which cells were stained with CMXRos alone and with a combination of DiOC 6 (3). They also noted that in fixed cells the retention of CMXRos depends on the amount of thiol level and that this may be a determinant of changes in CMXRos fluorescence. They concluded that further experimental work is needed to demonstrate the usefulness of CMXRos in measuring β¬βΏ m .
An extensive investigation of the properties of CMXRos has shown that the behavior of CMXRos, rhodamine 123, and JC-1 is similar in tests involving manipulation of the mitochondrial membrane potential (5). This is not to say that the fluorescence intensity obtained with any of these fluorescent lipophilic cations can simply be equated with the mitochondrial membrane potential. In particular, in a dynamic process such as apoptosis, the fluorescence from β¬βΏ m -sensitive dyes such as CMXRos may be influenced by factors other than the mitochondrial membrane potential.
First, the Nernst equation shows that, at thermodynamic equilibrium and under dilute conditions, the mitochondrial membrane potential changes inversely with the natural logarithm of the cation distribution. It stands to reason that during apoptosis or other forms of metabolic stress, the mitochondrion is not in thermodynamic equilibrium and that it does not confer ''dilute'' circumstances. The data shown in Figure 3 of Macho et al. (2) shows a saturation of fluorescence output as a function of dye concentration, which indicates that the requirement for dilute circumstances has been violated. Hence, in an intact cell system, the basic thermodynamic assumptions regarding the behavior of lipophilic cations around the mitochondrial membrane may not apply; therefore, the changes in fluorescence obtained may not accurately reflect changes in mitochondrial membrane potential. Thus, we (6) and Macho et al. (3) recommended using the lowest possible dye concentrations. Second, the DiOC 6 (3) stain may, as a result of the mitochondrial membrane potential, accumulate to such a high concentration that excimer formation and consequential red-shifting of fluorescence emission occurs. In fact, almost all dyes containing benzthiazole, benzoxazole, or benzimidazole moieties show a tendency