I want to detect apoptosis so, i used caspase 3 as a marker for detection. However, when i subject MSCs under serum starvation condition for 24 hours and detected caspase 3, i can only detect procaspase, but active caspase band was not detected. Interestingly, in the treatment group i found reduction in procaspase 3. Does this means that the treatment group had lower apoptosis or i should have to show active caspase 3 also.
Its very difficult to answer this question with out a little more information, Pro-Caspase-3 is inactive no matter what the quantity of expression, were your loading controls similar across control and treatment groups? I would be inclined to run a caspase-3 activity assay to confirm this finding, there are a number of colorimetric and fluorescent tagged assays on the market, qPCR analysis for caspase-3 would give you information about the expression pattern across the experimental groups. Essentially changes in pro-caspase-3 can not be used as markers of apoptosis.
Thanks a lot Andrew for your valuable and informative suggestions. your answer is appreciated
Honestly, I would look for cleaved-caspase 3 specifically. It is not a reliable measure to look at pro-caspase 3 expression only. you should look at cleaved-caspase 3 or cleaved-PARP levels atleast.