The use of chloromethyl-X-rosamine (Mitotracker red) to measure loss of mitochondrial membrane potential in apoptotic cells is incompatible with cell fixation.
BACKGROUND
A recent report by Macho et al. (Cytometry 25: 333-340, 1996) described the use of chloromethyl-X-rosamine (CMX-Ros) as a fixable probe for detection of loss of mitochondrial membrane potential (psi(mit)), an early event in many models of apoptosis. However, this previous report lacked a description of any direct comparisons between pre- and post-fixation analyses of normal and apoptotic cells stained with CMX-Ros.


METHODS
Using a variety of cell types, we investigated the effect of paraformaldehyde fixation on cellular retention of CMX-Ros and the implications of this for the subsequent analysis of changes in psi(mit) in cells undergoing apoptosis.


RESULTS
We found that following fixation, the resolution between normal cells with polarized mitochondria and apoptotic cells with depolarized mitochondria is reduced to the extent that accurate discrimination between the cell 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 applications for CMX-Ros in multiple parameter analysis of fixed cells are inappropriate and will lead to spurious results.
Gilmore
K
K
Department of Biological Sciences, University of Wollongong, Wollongong NSW, Australia.
Wilson
M
M
D017209:Apoptosis; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D005557:Formaldehyde; D018922:HL-60 Cells; D006801:Humans; D019169:Jurkat Cells; D008564:Membrane Potentials; D008928:Mitochondria / Q000737:chemistry / Q000502:physiology*; D009930:Organic Chemicals; D011108:Polymers; D016707:Tissue Fixation; D020298:U937 Cells
D005404:Fixatives; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011108:Polymers; C107472:red dye CMXRos; D005557:Formaldehyde; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C003043:paraform