Hello. I want to transient tranfect PANC-1 cells with a microRNA mimic with Lipofectamine2000 reagent in 6-well plates at a final culture volume of 2 mL/well. So, after platting cells and removing the media (with serum, no antibiotics), the microRNA mimic-lipid complex added to the cells must be in contact with them without any presence of serum and antibiotics, right? After 4-6 hours I remove the media and replace the wells with complete growth medium?
Thanks in advance.
Hello Alan,
we have experience with lipofectamine transfection of primary astrocytes. As you pointed out, during transfection we exclude serum and antibiotics. After 18h of adding the complex to the cells, we wash it with PBS and add medium with serum, but still without antibiotics. After 24h cells are ready for collection/regular cultivation. The conditions may work differently for your cells, but it works for us, we get about 30-50% transfection efficiency. Good luck.
Thank you very much, Ingrid Vargová.
One more question: in the well, can I mix the Opti-MEM medium with the regular medium (without serum and antibiotics)? Or it is better to only have Opti-MEM in the wells?
If your cells are sensitive to the medium change, I would use the base of the medium. If your cells are robust, I would stick to using only Opti-MEM or DMEM, because it increases transfection efficiency. Of course, remember to treat your untransfected control cells with this depleted medium. In the end, it all depends on the aims of your experiments, so you can do it even with full medium, it should also work to some extent. Hope this helps.
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