DiOC / PI question

Nebe-Von-Caron, G g.nebe-von-caron at unipath.com
Tue Apr 6 11:41:17 EST 2004


I would expect that to be quite normal as you measure the
premeabilisation of the plasma membrane and not of the mitochondrial
membrane. The mitochondria will loose their signal over time. I even
expect that you can have polarized bacteria that have permeabilized
membranes as long as they can pump faster than the influx comes through
the membrane. I like to borrow a bit from Howard's old book about
membrane integrity - e.g. the fact that you have a 21 g needle
permeabilising your blood vessel also doesn't make your heart stop, even
at a 9mm bullet hole it will still work for a while.
 You can actually use this as a energy run out experiment for your
mitochondria. Just stain them and then add a pore former to see how fast
they go. 

Regards

Gerhard


-----Original Message-----
From: James Marvin [mailto:jmarvin at flowcity.bsd.uchicago.edu] 
Sent: 02 April 2004 20:28
To: cyto-inbox
Subject: Re: DiOC / PI question


I've seen the same thing using MitoTracker Red (CMX Ros) and DAPI.
There 
is a population of cells with high MTR staining that's also positive for

DAPI.  I am also confused on how a cell can loose plasma membrane
integrity 
but still have a polarized mito.  I guess nonspecific binding, which all
of 
these mitochondrial dyes have to some extent could explain some of 
this.  Not sure though.

Marv



At 05:37 PM 4/1/2004, Richard K. Meister wrote:
>Greetings, all:
>
>I am running flow samples for an apoptosis assay using DiOC 
>(mitochondrial
>membrane potential) and PI (permiability).  Controls all look good and 
>"normal live" cells appear in the lower right quadrant of a PI(x) vs. 
>DiOC(y) histogram.  Necrotic cells appear in the upper left quadrant.
>
>However, while apoptotic cells generally appear in the lower left or 
>(dim
>PI) upper left quad, I also find in the experimental test samples about

>10% of the cells in the upper right quad.  I haven't yet been able to 
>explain the PI-dim(+) / DiOC(+) population.  Have any of you seen the
same 
>thing and/or have an explanation for what this population might be?
>
>Thanks in advance,
>Rick Meister
>
>
>
>
>
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