I am working on transfecting Ramos cells and currently using electroporation: the optimized protocol for the Amaxa Nucleofector system. The problem is I am not getting any luciferase readouts. I am using a Gaussia luciferase system; the luciferase is secreted into the media and cell lysis is not performed. I know the constructs work because they gave good results in Hek293 cells with Lipofectamine. I had a feeling the problem was detection sensitivity because I thought maybe the luciferase in the 1ml media for each well is too dilute therefore would go undetected. I tried incubating the cells in small volume of media (100ul) after electroporation but still can't detect anything. I have looked at different time points too and still nothing.
If anyone has some insight or an idea of something technically I might be doing wrong I'd be very happy to hear. As of now I am at a dead end with this. My colleague is doing transfections too using electroporation with THP1 cells and she is having the same problems as me and has also not yet detected any luciferase in the culture media.
Dear Justine,
If possible, I would advise you to use lentiviral vectors. We have nice transduction efficiency with our cell lines (erythroleukemic cells) (i.e. >90%). Lentiviral vectors with luciferase are commercially available.
Best,
Guillaume
Dear Justine,
Also you could try TurboFect Transfection reagent. In our lab we've obtained good results with transfection of primary human MSC.
But I think you should try to transfect the cells with other reporter gene (for example EGFP) in order to determine the transfection efficiency with your system. Because it might be the problem with poor secretion rate.
Best regards,
Ianina.
Hi,
Thanks for the suggestions, but a lentiviral system is not an option in our case. I will try a new transfection reagent called "X-treme Gene" There is a paper that involves coating plates with egg white or collagen to make the suspension cells adherent and they got good results using that transfection reagent and I think Turbofect as well http://www.biotechniques.com/rapiddispatches/High-efficiency-transfection-of-suspension-cell-lines/biotechniques-333908.html
hopefully the problem is not poor secretion rate, or that will be a major setback...
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