Is chloromethyl-X-Rosamine useful in measuring mitochondrial transmembrane potential?

The paper from Macho et al. (3) describes the suitability of chloromethyl-X-rosamine (CMXRos) as an aldehydefixable potential-sensitive fluorochrome to be used for the detection of mitochondrial transmembrane potential ( m ) changes during early apoptosis. In our opinion, the data presented in the paper do not fully support the interpretation that a straightforward correlation between CMXRos binding and m exists. In that Figure Treatment with mCICCP, an uncoupling agent that dissipates m , induces a reduction of CMXRos (and DiOC6) signal of more than 2 logs (from near 400 to near 2 channels of fluorescence). These data do not match those obtained in the single-color experiments described in Figure Thus, there is more than 1 log difference in the fluorescence signal generated by the same amount of CMXRos, depending on whether the latter is used alone or in combination with DiOC 6 . Such behavior suggests a spillover of the DiOC 6 ''green'' signal into the ''red'' channel in which the fluorescence of CMXRos is collected. In this light, much of the CMXRos fluorescence measured in double-color experiments would actually reflect DiOC 6 fluorescence and underlies the straight correlation seen between the two fluorochromes: dissipating m will reduce DiOC 6 -generated fluorescence, thereby rendering it no longer strong enough to produce the ''red'' signal.