more mitochondrial questions

Mark A. Miller mamiller at
Tue Sep 29 16:34:23 EST 1998

I should have added these questions:

Those of you who use mitochondrial potential dyes to monitor apoptosis:
do you wash the cells after loading with Rh123, DiOC6(3), MitoTracker,
JC-1, etc.?  My feeling has been that washing is the right way to go.
When the mitochondrial potential gradient collapses, the dye is free to
diffuse out of the mitochondria, but it could still be contained within
the cytoplasm, right?

Could nonyl acridine orange be useful in monitoring mitochondrial
dysfunction?  I was under the impression that NAO (as well as the
MitoTrackers) was an indicator of mitochondrial mass, not an indicator
of mitochondrial potential.  But I've also read that changes in NAO
fluorescence indicate changes in cardiolipin configuration.  Molecular
Probes also recommends MitoTracker Red CMX Ros as an indicator of
apoptosis?  What's the story there?

Finally, if you read my previous note:  When I stain cells with Rh123
and PI, I would expect the apoptotic cells to be Rh123 low / PI low.
However, when I view a scatter plot gated on Rh123 low / PI low, most of
the event are in the lower left-hand corner, just barely about the FS
threshold.  I would be inclined to call those events debris, or to be
generous apoptotic bodies/naked mitochondria.

There are also a very few Rh123 low / PI low events with slightly
increased FSC and slightly decreased SSC (relative to untreated  cells,
or even the "healthy" cells within the treated sample.)  Those are the
events that I would instinctively call apoptotic.  But the percentage of
these events doesn't change much from control cell to the treated

Mark A. Miller
University of Pennsylvania
School of Dental Medicine
Flow Cytometry Facility

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