I intend study the effect of TNF alpha on cancer cells especially myeloma by using proteomics approaches such as Mass spec, 2DE etc.
Are you using cell lines? Many MM cell lines do not respond well to TNF in our hands(proliferation).
But, for those that do: 10-20 ng/mL. Incubation time depending on what effect you want to look at.
Cells stimulated with TNF alpha secrete different types of cytokines, including TNF alpha. Basically this regulates the balance between propagation of inflammatory response and its eventual resolution. So, different cells respond differently and during the process of stimulation various cytokines are produced and several proteins interact, in order not to miss any of these cascade of events during elicitation, it is wiser to use a dose and time response. You might want to start with 10ng/ml as this gives an optimum response in many cell types and for cancer cells as Ms.Holien said, you have to increase the concentration as they are immune tolerant and remember not to use serum while inducing. Goodluck!
Hi Holien,
Yes,iam using MM cell line,and i will study the effect of TNF alpha on large sets of proteins,no specific target.And i need to study this effect on presebce and absence of one anticancer drug.I will do 2DE,so i do not prefer too much concentration and incubation points.
Hi Mulugu,
Thanks for your reply.As i said for Hoilen.I need to do screening look for the effects on different types of protein(proteomic study)so iam looking for the ideal point of incubation and concentration to keep the cell live and to be able to look for the changes in different sets of proteins
Hi Eric,
While stimulating cancer cell lines I usually try 30 ng/ml. the lenght of treatment also may depend on your proteins of interest. If they are related to TNF signaling, keep in mind that some of them get activated kind of fast so, depending on your focus 4h may be enough. You could also try up to 24h but no longer (specially if they are TNF sensitive).
Also, I recomend to leave the cells with no serum the night before the treatment and also during it.
Hope this helps! Good luck!
Hi,
If I want to induce necrosis in A549 (lung cancer cells), how long should I use TNFalpha for and what concentration?
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