Hello Ananya Chatterjee
Transfecting macrophages is especially difficult since macrophages are professional phagocytes that are very sensitive to foreign materials. Introduction of foreign DNA activates the Toll-like receptor 9 (TLR9) pathway leading to the production of cytokines and nitric oxide. These activated macrophages may then be less responsive to treatment that one may intend to examine.
You may need to optimize the protocol for various parameters such as timing of transfection, type of transfection reagents, amounts of transfection reagents and of plasmid DNA, as well as ratio of transfection reagent to plasmid DNA.
One critical step for RAW264.7 which you need to consider is the health of the cells. Overgrown cultures are not suitable for transfection as their physiology changes, and continuous culturing of RAW264.7 cells for a long period of time can also change the phenotype and function of the cells. Freshly thawed cells that have a low passage number are recommended for transfection.
Another important consideration is the choice of transfection reagents. Lipid-based transfection reagents are typically used in research due to its ease of use and commercial availability. The cells should be incubated with the transfection reagents for a few hours instead of overnight. Longer incubation time with the transfection reagent will increase transfection efficiency, but it can also be harmful to the cells either causing cell death or cell activation, both of which can interfere with the experimental design. Also, the time between transfection and experimental treatment (the rest time) is crucial.
You may want to refer to the paper attached below. It will be helpful!
Article Transfecting RAW264.7 Cells with a Luciferase Reporter Gene
Good Luck!
Yes they are easy to transfect with standard lipofectamin process. Here is the specific product tailored for macrophage transfection as it is important not only to transfect them but also not to activate them.
https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-raw267-3-macrophage-cells-using-lipofectamine-ltx-reagent.html
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