Fwd: Re: early apoptotic cells

Hi Sunny,

While what William has said is true, this may not be your problem if you do in 
fact see Annexin+/PI+ cells.  What we have found is that the detection of 
different stages of apoptosis is very time dependent.  Remember that you are 
just taking a snapshot of the cells at time of staining.  You most definitely 
need to do a time course with this assay to determine the appropriate time to 
look at what you want to see- if you look too late, you may not see early 
apoptosis but only the later stages.  For apoptosis studies, it is always good 
to do at least two assays (tunel, caspase activation etc) to confirm what you 
are seeing.  In our hands, we find the annexin assay the least useful since it 
is so fraught with caveats like the calcium dependence and time dependence.

Good luck!

Jonni



----- Forwarded message from William King <wking@aecom.yu.edu> -----
    Date: Tue, 13 Feb 2007 14:02:15 -0500
    From: William King <wking@aecom.yu.edu>
Reply-To: William King <wking@aecom.yu.edu>
 Subject: Re: early apoptotic cells
      To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>

Sunny,
Eric's comments are well said.
The binding of Annexin V to phosphatidylserine is calcium dependent  
and very tight with a Kd of approximately 5 x 10-10 M, consequently,  
your Annexin V buffer must have calcium in it to see Annexin V
binding. If you're using regular staining buffer or PBS to label your  
cells, you should not expect to see any Annexin V binding as regular  
staining buffer or PBS usually does not contain calcium.
Best,
William


Flow Cytometry Core Facility
Albert Einstein College of Medicine


On Feb 9, 2007, at 3:54 PM, Xiao Qun Zhai wrote:

> Hi,
>
> I am doing cell apoptotic/necrotic analysis using Annexin V/PI  
> staining method. Very interestedly, my samples do not show early  
> apoptotic population, which is Annexin V +ive/PI -ive. I am  
> wondering why? Is this related to cell type? I can't see any early  
> apoptotic cells in all my samples (control samples, high and low  
> drug concentration treated samples). Has anyone here met the same  
> problem? How to bring the early apoptotic population back?
>
> Many thanks
>
>
>
> Sunny Zhai
>
> Lancashire School of Health and Postgraduate Medicine
> Harrington Building
> University of Central Lancashire
> UCLAN
> Preston
> PR1 2HE
> UK


----- End forwarded message -----


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