RE: 7-AAD COMPENSATION PROBLEM

Dear Flower,
1. Did you used single-color tubes? If not, try to prepare a single-color
tube for each flurochrome you are using besides another tube for the
isotypes controls. 
2. On doing compensation with 7-AAD, try to use a log-scale instead of
linear. 
Greetings
Dr. M Elshal
Research Fellow
Flow Cytometry Core, 
NHLBI-NIH
Bethesda, MD
USA

-----Original Message-----
From: Kotsianidis, Ioannis [mailto:i.kotsianidis@imperial.ac.uk] 
Sent: Wednesday, January 26, 2005 10:29 AM
To: cyto-inbox
Subject: 7-AAD COMPENSATION PROBLEM



Dear Flowers,


I am trying to perform 7-AAD staining for cell cycle with simultaneous
surface staining.

When I tried to compensate 7-AAD (linear, FL3) with PE (log, FL2),


 1) I wasn't able to see any event on an FL2 vs FL3 plot, while running the
PE sample and 


 2) When I removed the FL3 signal out of FL2 while running the 7-AAD tube, I
lost all the PE positivity, 

both in FL1 vs FL2 plot and my final sample (stained with FITC,PE and
7-AAD).


I also observed the same phenomenon with APC when I tried to perform 3-color
analysis with 7-AAD.


Can someone please tell me what am I doing wrong? I've read the papers by
Rabinovitch (1986) and also Schmid (1991)

but they don't seem to worry at all about compensation problems...


Thanks in advance, 


Dr Ioannis Kotsianidis 
Clinical Research Fellow 
Department of Haematology 
Faculty of Medicine - ICSTM 
Hammersmith Hospital 
Du Cane Road 
LONDON W12 0NN 
U.K.