Hi,

I have been trying to use Ac-YVAD-cmk in an attempt to inhibit inflammasome activity. However, when running ELISAs for IL-1beta I am not seeing the inhibitory effect of the inhibitor. I have tried incubating with both 40um and 100um of the Ac-YVAD-cmk for 5 hours and then replacing the media before stimulating the cells with either the bacteria of interest or a positive control of 100ng/mL LPS and 5mM ATP (I have also done the experiments without replacing the media prior to stimulation). The Ac-YVAD-cmk is stored in -20C in DMSO. The literature indicates that the concentration and other facets of the protocol should be sufficient in inhibiting IL-1beta production. What am I doing wrong and what can I change to optimize this procedure.

Thanks in advance.