Has anybody transfected BJ and HEF cells with Lipofectamine 2000? If yes, what was your DNA:Lipo condition? I want to start optimization with a starting range for a 24 well plate.
Optimization for BJ cells is usually done by testing a variety of concentrations. I know this reagent (https://altogen.com/product/bj-transfection-reagent-skin-fibroblast-cells/) is pre-optimized for BJ cells with a protocol as well, so you might not need to test different concentrations if the cost of the consumables is a concern.
Rafal,
I did not use Lipofectamine 2000 but I have some experience with BJ's. These are primary cells that are very difficult to transfect with lipid based reagents. I used Mirrus LT1 reagent and was very unsuccessful. Generaly, in our lab we use electroporation method from Amaxa/Lonza to transfect BJ's or HFF's. However, one day I transfected a GFP plasmid provided as a control with electroporation kit using LT1 and I've got about 60% transfection efficiency...! Obviously, the key for effective liposome based transfection of primary fibroblasts is the quality of DNA you transfecting with. I asked the company what method of DNA isolation do they use for their GFP control plasmid, and they refused to tell me... The only suggestion they provided was that in the past they used some Qiagen Midi kit. Summarizing, I'm afraid that Lipofectamine wont be to effective in transfection of primary cells, but it may depend on quality of DNA that you are using.
Good luck!
Robert
Thank you Robert! I will have a look in literture waht people are doing...
For plasmid transfection of HEF I use Viromer YELLOW (from Lipocalyx).
Optimization for BJ cells is usually done by testing a variety of concentrations. I know this reagent (https://altogen.com/product/bj-transfection-reagent-skin-fibroblast-cells/) is pre-optimized for BJ cells with a protocol as well, so you might not need to test different concentrations if the cost of the consumables is a concern.
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