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.