Re: apoptosis assay for adherent cells

Eric Miller (
Fri, 13 Jun 1997 08:54:49 +0100 (BST)

I don't remember this particular issue being covered, but I certainly
have done this assay on adherent cells with no particular problems. The
crux of the matter appears to be maintaining the integrity of the cell
membrane as 1) phosphotidylserine residues are a normal membrane component
and will be available as soon as the membrane is disrupted by anything,
not just apoptosis and 2) Counterstaining with PI to see viable cells will
give misleading results if the cell membrane is compromised by the method
used to detach cells.
We use a commercial trypsin/EDTA mix to detach cells at 37 deg C
for 5-15 minutes depending on the line, neutralise with medium in 10%
serum and resuspend in whatever is needed for staining puposes. THis seems
to give reasonable results with annexin V. Our concern is that there is no
way of using stored material for this assay, as all storage and/or
fixation appears to disrupt the membrane.
Hope this helps.

Eric P Miller
Edinburgh Medical Oncology Unit

"Wouldn't it be nice if hospitals and schools had all the money they
needed and the army had to hold jumble sales to buy guns?"

On Thu, 12 Jun 1997, mcfarland david c wrote:

> I am sure there was a thread a while back on using annexin V with
> adherent cells. I browsed back through the mailing list archive and came
> up empty. I probably just missed it. I have a client that was asking
> about said protocol. Could someone point me in the right direction? Is
> there a publication on the subject?
> Thanks,
> David McFarland
> University of Illinois Biotechnology Center
> Flow Cytometry Facility
> PS This is aimed at the administrators of the list: Is there a way to
> do a search in the mailing list archives at the Purdue Cytometry
> website? Did I just miss that too?

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