Typically, we identify a cell line as “difficult to transfect” by using a positive control reporter, such as GFP or luciferase. Multiple delivery methods should also be tested for any new cell type and the most commonly and easy to use method is lipid mediated; however, if this approach doesn’t yield the desired results, electroporation can be performed using the same positive control plasmid. There are certain mechanisms of why specific cell types are hard to transfect. Some cell types, such as MCF7 or HepG2, prefer to grow in clumps or clusters which is not ideal for transfection because minimal membrane surface is exposed which compromises uptake; there are other cell types, such as blood or immune cells, that lack the proper endocytic machinery, which again can minimize uptake; and there are other cells, such as macrophages, that have an evolved uptake mechanism, but quickly breakdown and destroy endosomal contents.
The following steps can help with optimizing transfection for lipid mediated delivery with DNA or RNA, to improve the efficiency for a wide variety of cell types, even the difficult to transfect ones:
• Performing the recommended reagent protocol, with the indicated doses, will help to determine the "sweet spot" of the reagent for a particular cell type
• Optimizing for cell density so that the cells are 70-90% confluent on the day of transfection, can help to improve efficiency by almost 10-15%
• Maintaining healthy cells in log phase during sub-culturing
• Controlling cell passage number post thawing is important for the health of the cells; most cell types should be used between 4 and 25 passages for optimal transfection
• Using the recommended cell culture media