Annexin V

Help!


I have an investigator trying to do Annexin V staining and they are getting
some strange results.  I tried doing a search on this list and found an
awful lot about the protocol, but not the strange results they are getting.
I have attached a Power Point with two different samples (untreated and
treated).  When we load the sample, the Annexin V - FITC peak starts out
about 1500 stays there momentarily, then rapidly moves up the scale to a
peak around 11,000 then levels off.  If we load the sample, wait about 1
minute, then record, the peak stays steady at the higher level.  Also, we
observed if we loaded the sample, recorded (and got the changing signal) but
did not unload the tube, started acquisition again - the signal was at the
higher level and stayed steady (leading me to believe there is a
pressurization issue.)	The 7-AAD signal stayed relatively steady whichever
way we recorded.  After the recent discussion on the list, I did turn the
agitation off (temperature was off also) and there was no change.  We are
doing this experiment on a FACSAria.  We ran several different samples with
several different types and time-points of treatment with the same results. 


Questions:


1)	 What is causing the Annexin signal to rise?  

2)	We are also trying to test spleen cells for apoptosis (and getting
very similar results).	We do a quick cold water lyse to get rid of the red
cells.	Should this cause any problems for Annexin staining?  Any other lyse
options that are better or worse for this protocol?

3)	We are getting fairly high percentages of cells in apoptosis in our
untreated groups.  Any suggestions?

4)	Is the age of the buffer or Annexin reagent critical?


After a careful review with the technician, I found that they are staining
in a higher volume of buffer than recommended.	They are staining 200,000
cells in 200 ul of buffer.  They then add 5 ul of the Annexin - FITC,
incubate for 15 minutes in the dark, add 300 ul of buffer, then add 5 ul of
7-AAD and incubate another 10 minutes.	According the the Pharmingen
protocol they should be staining in 100 ul buffer, adding the Annexin and
7-AAD at the same time, incubating 15 minutes, then adding 400 ul of buffer.
Although I think they need to address these differences, I really don't
think they are contributing to the phenomenon we are seeing.  


I would appreciate any insight you can give me!


Thanks so much in advance.



Pam Shaw

Fluorescence Cytometry Core Facilitator

Center for Environmental Health Sciences

The University of Montana

32 Campus Dr, Skaggs Building 052

Missoula, MT 59812


Ph (406) 243-4974

email:	 <mailto:pamela.shaw@umontana.edu> pamela.shaw@umontana.edu

www.umt.edu/cehs <http://www.umt.edu/cehs/FluorCytomCore.htm>
/FluorCytomCore.htm





This attachment - 'Annexin V.ppt' -  148.48 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/aa7e26782203e8d84f4756c616465e368efbfde6.ppt