6-color on Partec CyFlow space: problems red channels

Dear Flowers,

This is a follow up of my mail from some days ago 
( http://www.cyto.purdue.edu/hmarchiv/current/1906.htm ). Yesterday I got BD's  
Compbeads and I tested six different fluorochomes with them: FITC,  
PE, PE-Cy5, PE-Alexa Fluor 700, PE-Cy7 and APC.
Unfortunately I got some strange results in some of the red channels  
which I can not explain.

In the PE-Alexa Fluor 700 channel (710/20) and PE-Cy7 channel (748/ 
LP) there is a very strange bimodal distribution of the  fluorescent  
intensity  of the beads. This is the same in the case of stained and  
unstaind beads ! This bimodality is the most prominent when I used  
CD8-FITC staining. The bimodal fluorescent distribution is the same  
with or without compnesation ( using FlowJo software ). You can see  
the images of the diagrams here:
http://pg.photos.yahoo.com/ph/almacsiga/album?.dir=/1651&.src=ph

Similar but not the same problem can be seen in the case of APC  
channel when PE-Cy5 staning was done alone. In this case after	
compensation only a portion of the crosstalking PE-Cy5 positive beads  
faded away from the APC channel, the others not, furthermore the  
beads which disappeared from the APC channel were overcompensated. I  
know that there is a significant cross-talking between PE-Cy5 and APC  
because the Cy5 in the tandem dye can be excited at 633 nm and Cy5  
emits in the APC channel which requires cross-laser compensation.  
Otherwise I don't understand clearly how cross-laser compensation  
works...
To my mind PE-Cy5 fluorescence should not be visible to the APC  
channel because of the time delay betwwen the 488 nm and 633 nm  
excitation so the fluorescence which come from the PE-Cy5 staining  
should be come from only the Cy5 part of the dye excited at 633 nm.  
If this theory is acceptable the Cy5 can not be compensated out from  
the APC channel, but still there is a portion tha fluorescence in the  
APC channel which can be removed.

This is a hard time for me to compile a working 6-color setup for the  
first time in my practice and I think this would be a big step	
forward comparing to the "simple 3-color world" of a BD FACSort.

Any advice is appreciated. Particularly I wonder the experience of  
the folks who work with Partec CyFlow space too.

Best regards,


Pal Jakso

University of Pecs
Faculty of Medicine
Dept. of Pathology
12. Szigeti str.
H-7643 Pecs, HUNGARY