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

✍ Scribed by Cristiano Ferlini; Giovanni Scambia; Andrea Fattorossi

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 16 KB
• Volume: 31
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19980101)31:1<74::aid-cyto10>3.0.co;2-v

✦ Synopsis

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 1, double-staining analysis indicates that 30 nM of CMXRos parallel DiOC 6generated fluorescence. 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 3A, showing that the highest achievable fluorescence signal with 30 nM CMXRos is near channel 20, and that it is reduced to near channel 1 upon mCiCCP treatment. 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.

The possibility that CMXRos fluorescence may not completely depend on ⌬⌿ m is also supported by the