Apoptosis again.....

medbury@renal.wsahs.nsw.gov.au medbury at renal.wsahs.nsw.gov.au
Tue Dec 23 07:42:36 EST 1997


From:          medbury at 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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