RE: Caspase activity in lymphocytes

Immunocytochemical detection of activated caspase-3 using Ab that reacts with the cleaved
(activated) form of this enzyme appears to be very sensitive and specific marker of its
activation. We have found that it correlates quite well with FLICA binding (Cytmetry,
55A; 50-60. 2003) and appears to be more sensitive and specific than the fluorogenic
substrate cleavage. As discussed in the above article, the nonspecific component in FLICA
binding, however, cannot be excluded.

Zbigniew Darzynkiewicz, M.D., Ph.D.




-----Original Message-----
From: Fredrik Thorén [mailto:fredrik.thoren@microbio.gu.se] 
Sent: Tuesday, September 21, 2004 5:48 AM
To: cyto-inbox
Subject: Caspase activity in lymphocytes


Hi all,
We are studying caspase activation in peripheral blood lymphocytes but have encountered a
problem. While a FLICA-assay (fluorochrome-conjugated
inhibitor) points to caspase activation, no caspase activation is seen using caspase-3
substrate-based assays on intact cells (Flow) or cell lysates (spectrophotometry).
 
Are FLICA assays more sensitive, or is the result from that assay possibly a false
positive due to unspecific binding?
 
What apoptosis inductor would you use as positive control for caspase activation in
non-cultured lymphocytes? 
 
Thanks in advance
 
/Fredrik