I used Lipofectamine 2000, 3000, PEI, Fugene 6 and HD, electroporation and no transfected cell. HELP!!!!
Yeah 3T3-L1 is a tough cell line to transfect. There are some pre-optimized 3T3-L1 kits (https://altogen.com/product/3t3-l1-transfection-reagent-emb-fibroblast-cells-cl-173/) that can give up to 70% transfection efficiency of siRNA. Larger plasmids can also result in lower transfection efficiencies, so using a complex condenser (in the kit above) can help you get higher nucleic acid uptake. Make sure you're following established protocols - you didn't write what protocol you're using, so we can't help you find any procedural errors.
Transfection in 3T3-L1 is definitely hard compared to other cell lines. I used Lipofectamine 2000 to transfect preadipocytes for 24 hours and then differentiated the cells for 6 days. The gene expression was reduced by 60%. Please be aware it's not 100% knockout, but it still allowed me to observe some differences at protein levels.
Also, I've seen some researchers keep transfected preadipocytes in stock. Once you have success with the cell transfection, you can do the same thing and have your own stock. Good luck!
Have you checked your cells for mycoplasma contamination? Mycoplasma can severely reduce transfection efficiency and is a very subtle type of contamination.
Based on my experience of tech support for the Viromer transfection reagents, these cells are very very hard to transfect. Nonetheless, there are users who had some success, in particular for siRNA transfection.
Please, have a look here
https://viromer-transfection.com/wp-content/uploads/2015/09/pre-adipocytes-and-adipocytes-transfection-viromer.pdf
and here
https://viromer-transfection.com/wp-content/uploads/NIH3T3-transfection-viromer.pdf
I transfected GFP to test the transfection and I saw like one cell transfected.
Yeah 3T3-L1 is a tough cell line to transfect. There are some pre-optimized 3T3-L1 kits (https://altogen.com/product/3t3-l1-transfection-reagent-emb-fibroblast-cells-cl-173/) that can give up to 70% transfection efficiency of siRNA. Larger plasmids can also result in lower transfection efficiencies, so using a complex condenser (in the kit above) can help you get higher nucleic acid uptake. Make sure you're following established protocols - you didn't write what protocol you're using, so we can't help you find any procedural errors.
Ted, thank you for your answer. I'm not using any specific protocol, I'm following what I found in some articles using 3T3-L1 and datasheet of the reagents. Ted Bremmer
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