I am working on a drug that is supposed to block NF-kB, so I want to study the effect of the drug on protein and mRNA expression in colorectal cancer cells. Should I induce NF-kB in these cell lines (using TNF-a as example)?
If yes, how I can use TNF-a to induce NF-kB? What is the protocol for adding TNF-a, and what concentration should I use and for how long? When I should add TNF-a to the cell (i.e. before or after drug treatment)?
Hi Bashir,
what I may do in your case is use as a positive control a condition in which you are sure you are inducing NFkB activation and then test your drugs and compare with your conditions.
I also work with colorectal cancer cells and when I stimulate then with TNF I use a concentration of 30 ng/ml for at least 24 h (just because I am interested on cell death induction). For NFkB activation I should activate with TNF 15-30 min, that would be enough for that (to be sure, you can check by WB the levels of IkBa so you may be sure about the best time as this differs from one cell line to another).
Moreover, I think that the best would be to stimulate with TNF before your drug treatment. The fact is for how long you are treating with your NFkB inhibitors as if it is too long and you are not adding TNF you might lose NFkB activation by TNF. on the other hand, if you plan to treat both with TNF and your drug for long times, you may be inducing apoptosis (also, keep in mind that TNF does not induce cell death in all cell lines. As far as I can tell, colon cancer cell lines behave differently after TNF treatment).
Hope this helps and good luck!!
Hi Bashir,
what I may do in your case is use as a positive control a condition in which you are sure you are inducing NFkB activation and then test your drugs and compare with your conditions.
I also work with colorectal cancer cells and when I stimulate then with TNF I use a concentration of 30 ng/ml for at least 24 h (just because I am interested on cell death induction). For NFkB activation I should activate with TNF 15-30 min, that would be enough for that (to be sure, you can check by WB the levels of IkBa so you may be sure about the best time as this differs from one cell line to another).
Moreover, I think that the best would be to stimulate with TNF before your drug treatment. The fact is for how long you are treating with your NFkB inhibitors as if it is too long and you are not adding TNF you might lose NFkB activation by TNF. on the other hand, if you plan to treat both with TNF and your drug for long times, you may be inducing apoptosis (also, keep in mind that TNF does not induce cell death in all cell lines. As far as I can tell, colon cancer cell lines behave differently after TNF treatment).
Hope this helps and good luck!!
Hi Lucia,
it is just as you said, you need a known activator of NFkB signaling pathway like TNF alpha or IL-beta. Secondaly, you have to construt a dose-respone and time cousre for the activator you select so as to select the best dose and the best time you achieve the most prominent activation of the NFkB pathway. Thirdly you preincubate your cell with the selected activator at the selected dose for the selected time befor you add your test drug
Thanks Dr.Bashier and Dr.Lucia for the valuable comments and suggestions, for sure it would help me a lot.
Dear Bashier,
just a correction to my answer you have to incubate the cell first with your drug, then without wash add TNFalpha for 30 minutes and thereafter harvest you cells and detect NFKb expression by WB
Best wishes
I am also use TNF alpha treatment for checking apoptosis and survival pathway. I do in HELA cells. So does anybody have experiences in this case?
Most of the colorectal cancer cell lines (like HCT116) are positive for TLR4. Why don't you try to trigger this receptor with LPS? You can get LPS very easily and I assure you that you'll see a nice NF-kB pathway activation as a result.
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