Authors' response: Chloromethyl-X-Rosamine?A fluorochrome for the determination of the mitochondrial transmembrane potential

Chloromethyl-X-rosamine (CMXRos) has been used successfully, alone or in combination with other FITC-labeled probes, to detect a disruption in the mitochondrial transmembrane potential (⌬⌿m) occurring in apoptosis.This probe has been used to demonstrate that, during programmed cell death, the ⌬⌿m breaks down before cells exhibit phosphatidylserine exposure (1).In these experiments, cells were labeled with CMXRos plus FITC conjugated to recombinant Annexin-V, and data were confirmed by parallel experiments involving DiOC6(3) (1).Moreover, CMXRos has been employed to show that the DYm is lost before cells undergo DNA fragmentation.In these latter experiments, cells were first labeled with CMXRos, then fixed with formaldehyde, and finally subjected to an FITC-dUTP-based TUNEL staining (8).Alternatively, cells were labeled with CMXRos and simultaneously stained with SYTO(R) nucleic acid stains allowing for the detection of nuclear DNA loss (6).Treatment of cells with respiratory inhibitors or uncoupling agents such as mClCCP or FCCP causes a decrease in CMXRos incorporation that is observed both in fixed and non-fixed cells (3,7), alone or in combination with other dyes (1,3,7,8).Although, in long-term experiments uncoupling agents such as mClCCP can provoke oxidative stress, we have shown, in an extensive series of control experiments, that mClCCP does not cause an increase in the generation of reactive oxygen species or a depletion of monochlorobiman-reactive thiols within the short term incubation (15 min) used to generate negative controls for CMXRos or DiOC6(3) stainings (2,4,9).Therefore, it appears clear that, in accord with its physicochemical characteristics, the lipophilic cation CMXRos incorporates into cells as a function of the transmembrane potential of the plasma and mitochondrial inner membranes.It is true that the difference in CMXRos-dependent fluorescence signals between control cells and mClCCPtreated cells decreases upon fixation (3,7), which may be due to the loss of CMXRos not interacting with thiols, perhaps due to the fact that CMXRos is in excess over the available thiols contained in the mitochondrial matrix (5).Thus, while there is no doubt on the usefulness of CMXRos for ⌬⌿m determinations, future amelioration in the fixation procedure and/or the generation of more potent CMXRos derivatives should be expected.