I am attempting to test cleaved caspase-3 activity after treatment with TNF-alpha at different time points. I am utilizing the Par-C10 epithelial cell line, and would like to know if anyone has experience doing this. I was planning on growing the cells in separate culture dishes for 24 hours in regular medium, removing the medium, and replacing it with regular medium with TNF-alpha mixed in. Would this be the best way to perform the experiment, or would serum free medium with TNF-alpha work best?
I wouldn't go for serum free since this could lead to starvation. You should try at least 1% FBS and make a "no cell" control to verify serum influence. Also be careful with antibiotics in your media, this can also give false positive results.
No experience with Par-C10 so not sure how sensitive it is to serum. If the cells are very sensitive to small amount of TNFa or other proapoptotic factor in serum, may need to serum starve or use very very low TNFa, otherwise the background apoptosis may be too high for you to discern the increase in caspase-3.
- Badges
- Science method