I have read that many researchers use 20 ng/ml for 15-30 minutes in other cancer cell lines, is this suitable for hepatic carcinoma?
I think it depends on the definition of the activation of NF-kappa B signal pathway.
1) Confirmation by Cytoplasmic/ Nuclear-Fractioned Western Blotting
If you consider nuclear accumulation of p65/p50 heterodimer as the activation of the signal pathway, the treatment with TNF-alpha at the concetration of 20 ng/ml for 15-30 minutes is appropriate.
2) Confirmation by Quantitative RT-PCR
However, if you would think the up-regulation of the target inflammatory molecules via p65/p50 heterodimer is the definition of the signal pathway, you have to treat for 3-6 hours.
I think it depends on the definition of the activation of NF-kappa B signal pathway.
1) Confirmation by Cytoplasmic/ Nuclear-Fractioned Western Blotting
If you consider nuclear accumulation of p65/p50 heterodimer as the activation of the signal pathway, the treatment with TNF-alpha at the concetration of 20 ng/ml for 15-30 minutes is appropriate.
2) Confirmation by Quantitative RT-PCR
However, if you would think the up-regulation of the target inflammatory molecules via p65/p50 heterodimer is the definition of the signal pathway, you have to treat for 3-6 hours.
Most cancer cell lines have relatively higher constitutive transcriptional activity of NF-kB. Therefore, further stimulation of the NF-kB activity in certain cancer cell lines requires a much stronger stimulus. Depending upon the measure that is used to assess the stimulation of the NF-kB activity, one needs to optimize both the concentration of TNF-alpha and time for a desirable outcome.
I agree with Dr Divaker Choubey's opinion. Surely HepG2 cells do not have the constitutive active mutation in NF-kappa B signaling pathway, but they need to be treated with higher concentration than your experimental design. For instance, HepG2 cells were treated with TNF-α (100 ng/ml) or the vehicle for 12 hours.
Ref; Am J Physiol Cell Physiol. 2001 Jun;280(6):C1636-44.
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