And normally also, how to increase the efficiency of transduction in endothelial cells as I am getting a very low efficiency. I have used Lipofectin with 80% confluency.
You could try Turbofect, which is cheap and can even transfect senescent and differentiated cells with good results. Alternativly Matra, a magnet assisted transfection system.
Have you made a standard range with different concentrations of lipofectamin and DNA (or siRNA) ?. Personnaly I used a siRNA-Al488 to verify the rate of transaction and select the best combination.
On many cell lines, it is recommanded to apply transfection with less than 60% confluency.
Finally, I have best results with electroporation, but you will loose 50% of cells, but the other 50% could be positive for you insert.
Sylvain... i understand about the confluency but do you transfect when cells are seeded onto the culture insert. Its on a semi permeable membrane........ so how do i go about it?
Yes, I use OPTI-MEM-I to mix with transfection reagent and plasmid. But, for some reason some siRNA or plasmids just have low trandfection efficiency although I use the same primary cell culture and same transfecting reagent. Whatever work for you is the best protocol for you. Mostly I just keep trying. I have used lifopefctamine 2000, 3000, and LTX for plasmid and RNAiMAX for siRNA. I used reverse transfection method which requires less plasmid or siRNA.
Thank you for your answers. But I am still not getting the right clue to go about with transfecting on cells seeded onto a cell culture insert for permeability assay.
THe best tool is, whenever you have the tool, an adenovirus...you will get nearly 100 % of transfected cells at a MOI of 10
Hi,
As Gerold Untergasser suggested better option would be lentiviral vectors. Well I dont know about your experimental plan, but if your gene of interest on inhibition compromises cell viablity or induces rapid changes in cell physiology, try inducible vectors. IV will give you flexibilty to induce gene inhibition for any duration and at any surface, even in suspension.
http://www.clontech.com/US/Products/Inducible_Systems/Tetracycline-Inducible_Expression/Tet-On_3G
Best
Shenha,
maybe the attached publications might be helpfull. The reagent is available via the attached link
www.synvolux.nl
I transfect 80% confluent endothelial monolayers with Lipofectamin2000 in Opti-MEM and not in their culture medium for 5,30 h. After that time i remove the Opti-MEM and I relace it with normal culture medium without antibiotics.
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