I recently purchased a Caspase 3/7 Glo assay kit from Promega for apoptosis detection.My assay was done with HEK293 cells which were stimulated with Etoposide.I performed the assay exactly described in the protocol,and detected the luminescence with our Luminoskan Ascent luminometer. Unfortunately, in my assay I observed no induction of caspase activity and that the highest reading was equal to the "vehicle control".To rule out the problems with the system I also tested the same kit with purified caspase 3 in different amounts and observed the same outcome.I use Corning Costar 3610 96 well plates and seal the bottom of my wells with white opaque vinyl sealing tapes.
(I have played with different concentrations of etoposide,different incubation times,signal intergration times,different voltages of the luminometer PMT,DMSO and glycerol as solvents which are "ok" according to the protocol.I have also tried to place samples far away in the plate to avoid the possibility of crosstalk.)
Has anyone faced a similiar problem?Any suggestions what might be going wrong??
P.S.I have attached some data files.