Our computational work identified two natural compounds to be probable inhibitors of caspase-3. What are the types of cells that can be used for this experiment which involve apoptosis-induced cells as starting requirement. Whether I can use any animal/human cells (nucleated) to perform this experiment or need specialized cells? Also indicate which one assay kit is suitable for me?
http://www.biovision.com/caspase-3-colorimetric-assay-kit-3478.html
OR
https://www.caymanchem.com/product/10009135
(No offence on proprietary products, All rights reserved with them!!!)
Hi,
You can find a lot of information online about expression of caspases in many different cell lines. HeLa cells, for instance, express caspase-3. Just look for the type of cell line that is better for you (cancer cell lines, immune cells, etc)
In order to induce caspase-3-dependent apoptosis you can treat the cells with different compounds, like those listed here:
http://www.scbt.com/chemicals-table-caspase-3_activators.html
I have also used Staurosporine and it worked just fine for me.
Using some of these activators you can test whether your compounds inhibit caspase-3 activation or not.
I have detected caspase-3 activity using the fluorometric caspase-3 assay kit (Abcam), and also detected cleaved caspase-3 by Western blot (primary antibody Cell Signaling #9662), and got good results.
Hope this helps!
As separate data set of for your experiment, I suggest treating HeLa cells with your inhibitor and TNFa stimulation, then perform Annexin V\PI you will se a result as TNFa will induce Caspase dependent apoptosis in cells that shuld be detected by Annexin V positive population. If you drug work you supposed to see less cell death in cultures treated with you inhibitor.
Just a little bit of follow up to my last answer if you are really going to do HeLa TNFa survival assay in order to get better results you need to obtain Rip3 k.o. cell line, since in presence of caspase inhibitors cells will still go to Complex II Rip1-Rip3 cell death that might affect your data with RIP3 k.o. the only caspase dependant Complex I cell death pathway will be left working thus producing drastic difference in cell survival in your experiment.
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