Chloromethyl-X-rosamine is an aldehyde-fixable potential-sensitive fluorochrome for the detection of early apoptosis
✍ Scribed by Antonio Macho; Didier Decaudin; Maria Castedo; Tamara Hirsch; Santos A. Susin; Naoufal Zamzami; Guido Kroemer
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 715 KB
- Volume
- 25
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
Early apoptosis is invariably accompanied by a disruption of inner mitochondrial transmembrane potential (AP,). Cationic lipophilic fluorochromes, such as 3,3 ' dihexyloxacarbocyanine iodide (DiOC6(3)), rhodamine 123, or 5,5',6,6'-tetrachloro-1,l' ,3,3'-tetraethylbenzimidazolcarbocyanine iodide UC-l), can be used to measure such an apoptotic AP, dissipation. However, these dyes are afflicted by the handicap that cytofluorometric anal- yses must be performed ad hoc on nonfixed, metabolically active cells. Here, we show that chloromethyl-X-rosamine (-0s) is a viable alternative to other AP,-sensitive probes, and that it allows for formaldehyde fixation of cells before analysis. Using this fluoiochrome, 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 APm. In addition, we developed an approach to assess simultaneously APv, 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, ClWLRos provides several methodological advantages over other, nonfbrable fluomchromes used for A%, determination. o 19% ~lley-~iss, hc.