Promega Cytotox kit

[CARMEN ELLIOT]

I spoke with Promega's tech-help.  They have heard many cases where primary (
freshly explanted) splenocytes showed high spontaneous LDH release.

Their suggestions were to decrease the time of incubation (effectors vs 
targets in vitro) and decrease the time of substrate conversion by the 
enzyme (dark incubation of supernatants and substrate mix).  I've tried this 
with some success.  I'm measuring CTL response between cells with MHC 
mutations.  I don't expect a whopping response because of the minor 
differences, and I'm not certain that the slight response I see with this 
kit is due to CTLs or assay-difficulties.

If anyone knows if this or any other nonradioactive CTL protocol (Molecular 
Probes has a live-dead fluorescent kit) that is reliable for small 
differences in response, please let me know!

Thanks in advance.
Carmen