4 replies to this topic

[kaposi_pc]

Hello all,

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.

Attached Files

•  caspase measurement data.xls   27K   4 downloads
•  eto_titr_8hr.xls   36.5K   4 downloads

[KevinK]

Hello Kaposi_PC,
   Can you elaborate on some of the different incubation times you have tested?  I have done some looking into treatment times for etoposide and they seem to vary widely.  We have used it in-house with Jurkat cells at 6 hrs and had good results, but other publications show caspase cleavage after 24 hours but not at 16 in HEK293.

I am checking in with our R&D scientist to see what his eperience and suggestions may be.  If I can be of any assistance in the meantime, please feel free to contact me here or contact Promega Technical Services.

Regards,
Kevin

[kaposi_pc]

Hello Kevin!

Thanks for the reply.With incubation times,do you mean the ones after stimulation with Etoposide or Glo reagent+cells? I have made measurements 1hr after addition of Glo reagent to the cells.I have tested 2,4,8 and 24hrs for Etoposide stimulation.

Meanwhile I was in contact with the tech support and I was suggested to let the Glo buffer be at room temp for half an hour before using it.I did so and used the buffer for measuring purified Caspase 3 activity and it looks much better than my previous experiment (see attached).

With regard to the Etoposide experiment,please let me know if there are any updates on incubation time and concentrations for Etoposide stimulation of HEK293 cells.Also would be great to know if you have tested other reagents to induce p53 mediated apoptosis in HEK293 cells.

Thanks a lot!!!

[kaposi_pc]

Here's the Excel file!

Attached Files

•  Caspase3evaluation.xls   15K   7 downloads

[enen]

hello kaposi-PC

I did siminar experiment with you. I used etoposide to treat my cells for different time,and i also use different concentration.I also use the caspase 3/7 glo assay kit from promega.

I got good results. BUT  BUT last week ,the old kit was used out,I use the new kit that the LOT number is different from the old one, I can not get any differnt signal between treated cells and the control cells.  It is very stranger,why? could you tell how about you experiment.  Is it possible that the new kit didinot work well?

who can tell me   help me .


thanks a lot

Edited by enen, 15 March 2012 - 02:21 AM.