We used a endotoxin free kit to prepare our plasmids. But we found IFNα proteins could not trigger IFN-dependent reporter genes in A549 cells when compared to mock treatment. We therefore postulate maybe transfection of reporter plasmids already triggered IFN response to a maximal level in A549 cells, then the IFNα treatment did not effect any more. It is only a guess.
Hi!
(if I understand the question correctly) I have some suggestions.
Are you sure that IFNα isn't degraded?
The IFN-luc plasmid is working properly?
Are you sure that A549 cells are responsive to IFNα? Or are able to activate IRF3? (normally all cell type are responsive to IFNs, but A549 are tumorigenic cells, so can happen that IFN-R molecular pathway does not work)
Try a more physiological cell line (293 for example) + IFN-luc + IFNα
Furthermore, can you test if is there a mycoplasma contamination? mycoplasma can triggered IFN response to a maximal level.
Moreover, do you use liposomes transfection? Try different lower quantity of liposomes. High levels of liposomes can induces toxicity and induce IFN production.
Good Luck.
Bye!!!
Hi Domenico,
we used fresh IFNα aliquotes -but of cause we don't know if they were degraded in the cell culture medium for 24 h treatment, and they have some effects in 293T cells. According to literature, A549 cells should respond well to IFNα treatment. It's one of the standard cell lines used for determining the bioactivity of IFNα products. Someone did very similar experiment in published papers. So we do not know what happened. Liposomes should be a factor to be considered. Thanks!
Hi, I think these two articles below may answer your question well,
http://www.nature.com/ng/journal/v34/n3/full/ng1173.html
http://www.nature.com/mt/journal/v11/n1/full/mt200515a.html
Yeah, you can try. My data before showed that plamids(with or without inserts) transfection can induce ISG15 expression in HeLa cells. Good luck!
Hello Han Xiao,
I appreciate if you could tell me if you arrive to some explication about your results, because I´m thinking that the transfection with a DNA plasmid (with or without insert of interest) with a cationic liposomal reagent elicits interferon and prevents the infection with DENV 24 h post transfection, but the control without transfection has been infected.
Thank you very much
I'm having a similar problem where I am transfecting cells with a plasmid containing a luciferase gene preceded by an IFN-driven promoter. I have a high level of background expression in my untreated control (no IFN-b) and IFN-b is not inducing higher expression. I'm thinking the transfection is inducing IFN-I secretion, which could make sense given that cytosolic DNA is known to induce IFN-I via the STING pathway, in particular in immune cells (I'm using a macrophage cell line)...although I havent confirmed it experimentally. Does anyone have any thoughts on this?
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