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 mit ochondrial transmembrane potential (Delta Psi(m)). Cationic lipophilic fluorochromes, such as 3,3' dihexyloxacarbocyanine iodide (DiOC(6)C3) ), rhodamine 123, or hloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanin e iodide JC-1), can be used to measure such an apoptotic Delta Psi(m) dissipation. However, these dyes are afflicted by the handicap that cy tofluorometric analyses must be performed ad hoc on nonfixed, metaboli cally active cells. Here, we show that chloromethyl-X-rosamine (CMXRos ) is a viable alternative to other Delta Psi(m)-sensitive probes, and that it allows for formaldehyde fixation of cells before analysis. Usi ng 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-s urface antigens, and CMXRos is used to measure Delta Psi(m). In additi on, we developed an approach to assess simultaneously Delta Psi(m) and expression of intracellular antigens. Thus, the expression of Bcl-2, a mitochondrial outer-membrane protein, can be determined after staini ng with CMXRos, fixation, and cell permeabilization. CMXRos labeling c an also be combined with determination of apoptotic DNA fragmentation using the Tunel technique. In conclusion, CMXRos provides several meth odological advantages over other, nonfixable fluorochromes used for De lta Psi(m) determination. (C) 1996 Wiley-Liss, Inc.