I am trying to validate TNF-alpha inhibitors using nHDF (Human Dermal Fibroblast). According to the literature protocol, ActinomycinD (ActD) is applied to block the transcription followed by TNF-alpha. Both of which collectively will lead to cell death but none of them alone. I usually apply TNF-alpha after 1hour of ActD treatment. Then after 24hrs, I do MTT assay. Sometimes, it kills the cells significantly, sometimes not and sometimes non-significantly. I am just wandering that why it is so? Is it because of insufficient level of caspases because of ActD? Should I apply TNF-alpha first? Should I increase time between treatments? Any suggestions will be highly appreciated.
Hello Nasir,
the idea is that actinomycin D would prevent the upregulation of NF-kB induced cell survival proteins after TNF stimulation. You might want to check first whether the concentration of ActD used is sufficient to effectively block transcription. In my experience, ActD already has considerable fibroblast cytotoxicity on its own so you might not see an additional effect of TNF. For this reason, I prefer to use cycloheximide, a general protein translation inhibitor (250-500ng/ml).
best,
Yves
Dear Yves,
Thanks for your suggestion. In my case, TNF-alpha shows non-consistent behavior which is driving me crazy. ActD is showing around 20% cytotoxicity in my case which increases or remains same on applying 1ng/ml recombinant human TNF-alpha. This non-consistent level is creating hurdles for me to optimize the conditions for subsequent experiments.
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