I've been trying for several weeks to get consistent and successful transient transfection of glosensor DNA (promega) into Sw620 and Sw480 colon cancer cells in order to measure cAMP levels by luminescence.
I've tried both electroporation and lipofectamine 2000 with the latter giving better results but still yielding low transfection efficency (measured by GFP transfection - 20% for 620s and 40% 480s).
When I do get 'successful' transfection, it's in the 480s which appear less resistant to transfection but results are inconsistent when generating a forskolin/ibmx-induced stimulation of cAMP levels.
Does anybody have advice about transfecting these cells or hace suggestions for other methods to measure intracellular cAMP so I can study potential agonist-induced inhibition of cAMP production by these cells?
Thanks
Hi,
According to studies on SW620 and SW480 cell lines, one of the best methods for transfection is to use polymer-based transfection kits. So you can use Polyethylenimine (PEI) or commercial kits such as Arrest-In reagent or JetPei.
We've found lipofectamine 3000 to be an improvement in some of our difficult to transfect primary cells. Might be worth trying. Other thing that could help is to transfect your cells in suspension with the lipid complexes before seeding cells into the plate. Reverse transfection it's referred to, although it's not actually reverse.
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