The use of chloromethyl-X-rosamine (Mitotracker red) to measure loss of mitochondrial membrane potential in apoptotic cells is incompatible with cellfixation

Background: A recent report by Macho et al. (Cytometry 25: 333-340, 1996) d escribed the use of chloromethyl-X-rosamine (CMX-Ros) as a fixable probe fo r detection of loss of mitochondrial membrane potential (psi(mit)), an earl y event in many models of apoptosis. However, this previous report lacked a description of any direct comparisons between pre- and post-fixation analy ses of normal and apoptotic cells stained with CMX-Ros. Methods: Using a variety of cell types, we investigated the effect of paraf ormaldehyde fixation on cellular retention of CMX-Ros and the implications of this for the subsequent analysis of changes in psi(mit), in cells underg oing apoptosis. Results: We found that following fixation, the resolution between normal ce lls with polarized mitochondria and apoptotic cells with depolarized mitoch ondria is reduced to the extent that accurate discrimination between the te ll types is no longer possible. Conclusions: Overall, our results are consistent with CMX-Ros being a valid probe for psi(mit) in intact cells but only when the cells are stained and analyzed immediately. Thus, our results suggest that the proposed applicat ions for CMX-Ros in multiple parameter analysis of fixed cells are inapprop riate and will lead to spurious results. (C) 1999 Wiley-Liss, Inc.