Re[2]: Apoptosis again.....

From: Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
Date: Tue Jan 06 1998 - 14:01:49 EST


          Hi Heather and a happy new year to everyone!
          
          Unfortunately you do not specify the fixation method 
          (concentration / time and temperature) and whether your 
          method permeabilised the cells (PI or 7AAD uptake). We found 
          the loss of fluorescence with fixation time to be a general 
          matter, more pronounced with PE than FITC, perhaps due to 
          protein / antibody denaturation. You also have to be careful 
          of the final pH of the solution as 0.1 or 0.2 pH can make 
          quite some difference.
          To find out whether the fixation causes the fluorescence to 
          decrease or the longer incubation gives increased binding 
          (or both) samples would need to be fixed and measured at 
          different time points.
          The crunch question: what did they look like next day?
          
          Gerhard


______________________________ Reply Separator _________________________________
Subject: Re: Apoptosis again.....
Author:  medbury@renal.wsahs.nsw.gov.au at INTERNET
Date:    24/12/97 14:16


From:          medbury@renal.wsahs.nsw.gov.au
Organization:  Renal Medicine, Westmead Hospital.
To: cyto-inbox
Date:          Fri, 19 Dec 1997 13:01:26 EST+1000
Subject:       Apoptosis again.....
Priority:      normal


I set out to answer my own questions from 19/12/97
so here are my questions, with my answers

Hi,
 I have started doing annexin V binding (clontech) to monitor
apoptosis in endothelial cells. I have a couple of questions

I am getting what looks like non specific binding, a slight shift to
the right by the main population (compared to the no ab control),
separate to my specific peak which is further to the right. Any ideas
why? Does the use of Trypsin EDTA affect the cells and alter there
binding pattern? Or is it perculiar to EC's

The non specific binding was perculiar not to EC's but to the
antibody I was using. Until my antibody came in I was using another
guys antibody. When mine came in I compared the two. I only got the
non specific binding with his antibody. Both were clontech. Dont know
why the difference between the two.


I am taking the cells at different time points, ie at 6 hours and at
10 hours. I was fixing the cells because in the annexin binding
method you dont wash away excess antibody. The cells would be
sitting around till the next day, and I thought that they would be
slowly dying and reacting with the antibody.
Is fixing the cells okay? will they continue to react with the ab if
not fixed?

Okay so I compared fixing with non fixing. I took cells at 8hrs,
added the antibody. After 15min I fixed tube A but didnt fix tube B
It was 1hr before the tubes were analysed on the facs.
THe result was that tube A and tube B both gave a specific peak of
about the same height, however, the specific peak for the fixed tube
A was not as far to the right as the unfixed tube B. I tested two
different samples with the fixing and unfixing and the result was the
same.
If fixation opens the cells and exposes more PS you would expect the
shift to be further to the right, but it wasn't.
If in the unfixed tube more cells were dying with the extended time
and  putting PS on their surface and therefore binding the antibody,
then you would expect a higher peak for the unfixed cells, but there
wasn't.
 It suggests that in fixation of cells, there is no change in
the number of cells binding, but a decrease in the intesity of the
fluorescence.

Hope this helps others who have the same questions
further comments are welcome

Merry Christmas
Christ was born to save.



Heather
Heather Medbury PhD
Department of Surgery
Westmead Hospital
Westmead, NSW, 2145
Australia
ph (612) 9845 7680
fx (612) 9893 7440
Heather Medbury PhD
Department of Surgery
Westmead Hospital
Westmead, NSW, 2145
Australia
ph (612) 9845 7680
fx (612) 9893 7440



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