To simulate inflammation under In Vitro condition, I'm using DMEM (10%FCS+1%Pen/Srp) combined with 10ng/ml IL-6, TNF alpha and IL-1 beta. Due to the complexity of inflammation and role of different cytokines and chemokines, I'm not sure my protocol contains enough inflammatory substances.
Hi Amir,
Incubated your cells in the absence or presence of LPS (5 ug/ml) during 18 hours (5% CO2 and 37ºC).
Dear Amir,
I agree with your protocol based on pro-inflammatory cytokines and also believe that addition of LPS is a good idea.
Good luck!
There is a big problem! Human derived fibroblast cells, the cells which I use for my expriment, do not express CD14 (LPSR) and prpbably LPS doesn't have inflammatory effect!
What do you think about oder pro-apoptotic factors like Staurosporine?
TNF and IL-1b together should generate a strong inflammatory stimulus. Be careful with using LPS though, it can easily contaminate everything within a biosafety (flow) hood. For endothelial cells, either TNF or IL-1b alone works well and are widely used as in vitro models for inflammation.
Dear Subhadeep,
Thank you for your answer.
Im agree that IL-1b and TNF are the best pro-inflammatory cytokines, and they provide easy way to apply the inflammation under In Vitro condition, but they are not enough! as you know natural inflammation contains pro- and anti-apoptotic cytokines. The cocktail of IL-6, IL-1b, TNF, IL-10 and TGF could be effective but Im not sure about their half life in medium.
What do you think about the co-culture (fibroblasts+ Macrophages) ?!
Macrophages can be easily activated by LPS. Another option is to use the chemotactic formylated tri-peptide fMLP.
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