I have been trying to transfect P-cadherin GFP plasmids into previously generated knockout MCF10A cells (passage no. ~40) but have not been able to get the cells to show GFP.
I use NEON transfection 100uL with 500K cells with the following parameters: 1650V, width 20ms and 1 pulse. These settings were used for the knockout process previously and also in similar rescue projects which had shown positive results.
Would like to ask if anyone had done transfection on such old cells before and succeeded as I won't be able to generate any younger cells and if there are any suggestions which may help with the project. Thank you.
We use RiboCellin for siRNA knockdown transfection and Lipofectamine 3000 for plasmid DNA transfection and they both work really well, but I have only tried up to passage 20 after thawing.
I find MCF10As also seem to be really sensitive to transfection of any kind, so my best results often come when the cells are almost overconfluent when transfected. That said, I have definitely been able to express GFP and a number of other GFP-tagged proteins in MCF10A using 1.25ug plasmid DNA and Lipofectamine 3000 at any passage.
Perhaps try higher initial number of cells or a different transfection method? Good luck!!
I too used MCF10A for transfection with GFP-tagged protein using Lipofectamine. It works well.
Article p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Moti...
This paper may help you.
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