Chloromethyl-X-rosamine is an aldehyde-fixable potential-sensitive fluorochrome for the detection of early apoptosis

Early apoptosis is invariably accompanied by a disruption of inner mitochondrial transmembrane potential (ΔΨ(m)). Cationic lipophilic fluorochromes, such as 3,3' dihexyloxacarbocyanine iodide (DIOC₆(3)), rhodamine 123, or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), can be used to measure such an apoptotic ΔΨ(m) dissipation. However, these dyes are afflicted by the handicap that cytofluorometric analyses must be performed ad hoc on nonfixed, metabolically active cells. Here, we show that chloromethyl-X-rosamine (CMXRos) is a viable alternative to other ΔΨ(m)-sensitive probes, and that it allows for formaldehyde fixation of cells before analysis. Using this fluorochrome, we developed a three-color staining technique in which two fluorochromes (fluorescein isothiocyanate and phycoerythrin) coupled to antibodies are employed to determine expression of cell-surface antigens, and CMXRos is used to measure ΔΨ(m). In addition, we developed an approach to assess simultaneously ΔΨ(m), and expression of intracellular antigens. Thus, the expression of Bcl-2, a mitochondrial outer-membrane protein, can be determined after staining with CMXRos, fixation, and cell permeabilization. CMXRos labeling can also be combined with determination of apoptotic DNA fragmentation using the Tunel technique. In conclusion, CMXRos provides several methodological advantages over other, nonfixable fluorochromes used for ΔΨ(m) determination.