I transfected A549 cells with IFN signaling reporter plasmids( ISRE/IFNβ/IRF3-firefly luciferase) and CMV-renilla luciferase. 6h later 10,000/1000/100/10 U/ml IFNα2a or IFNα2b proteins were added to the cell culture. Then 24h later, dual luciferase assays were performed. However, it shows no specific induction for the reporter activities by IFNα. The A549 cell line was newly bought from Sigma (ECACC source). According to literature they should respond well to IFNα treatment---actually it is the standard cell line used for determining bioactivities of IFN proteins. And the IFNα products were produced by PBL Interferon Source and distributed by Invitrogen. Could anybody help me to analyze the reason? Thanks!
It sounds like your luciferase reporter is not functional or did not get into your cells. The transfection reagent GenJet claims a high (78%) transfection efficiency for A549 cells, so maybe that can help you.
Dear Mark and Kevin,
thanks for your answers! It seems my respond was not uploaded last time by laptop problem. Well, as respond to your suggestions, yes, I could reach a transfection efficiency over 50% for the A549 cells by using Lipofectamin LTX reagent. And I controlled the experiment by cotransfection of an EGFP expression plasmid. For the IFN-dependent reporter plasmids, I could get nice response when we cotransfected the HEK293T with the reporters and a constitutively active RIG-I expression plasmid.
Right now we actually guess it might be a kinetic effect , or maybe the A549 cells were already activated by transfection of DNA---the IFN responses were already triggered to a maximal level by foreign DNAs, then the IFN proteins could not increase the reporter activities any more. So I would like to raise another question, does anyone knows if transfection of DNA could trigger IFN response in A549 cells?
I am looking forward to hearing from you.
Han
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology