CFSE part 2

Oops, forgot to add the following.

I was wondering if it was possible to use a FL2 fluorochrome along with
APC and PerCP on CFSE labelled cells. If CFSE leaks into the FL2 channel
- is it nearly impossible to use a PE or Cy5-PE conjugate? If someone
has successfully done this, could they let me know what tricks they
used?

I hope everyone understands what I'm trying to do.

Thanks

Matthew Henry

CD45 Group
Edward Jenner Institute for Vaccine Research
UK

-----Original Message-----
From: Matthew Henry 
Sent: 05 May 2004 16:46
To: cyto-inbox
Subject: CFSE

Hi Flowers

I'm trying to set up a three colour CFSE experiment on a FACScalibur and
am relatively new to flow so please excuse any dumb Qs.

I've set up my CFSE-labelled cells with PerCP and APC conjugated
antibodies. The compensation between FL1 and FL2 is massive (~95%)... is
this normal, and can I get round this, other than using the lowest
concentration of CFSE (yes, I have titrated it already 1.25-2.5 uM CFSE
seems to work best)? 

There also seems to be some spillover from the CFSE into FL3 channel -
this cannot be compensated for on a BD FACScalibur - do you just have to
live with this or can it be resolved? If CFSE is a FITC derivative, I'd
have thought that it should not spill over into the FL3 channel - am I
correct in assuming this or is something happening which I don't know
about?


Thanks

Matthew Henry

CD45 Group
Edward Jenner Institute for Vaccine Research
UK