Transfecting Primary Macrophages with Modified mRNA

Prepare for transfection by adding the pre-calculated volume of mRNA transfection buffer minus the volumes of mRNA transfection reagent and the mRNA to a reaction tube. Thaw the mRNA stock and mix it gently by flipping the tube. Then, add the pre-calculated volume of mRNA to the reaction tube.

Vortex and spin down the reaction mix and the mRNA transfection reagent, then add the appropriate volume of the mRNA transfection reagent to the reaction mix tube. Vortex the tube, and spin it down, then incubate it at room temperature for 15 minutes. Meanwhile, replace the culture medium of the macrophages with fresh, warm culture medium.

Once incubation of the transfection mix is complete, add the mix to the plate wells containing the macrophages dropwise and in a circle from the outside to the middle of the well. To ensure uniform distribution of the transfection mix, gently rock the plate in a vertical and horizontal direction.

Then incubate the plate at 37 degrees Celsius and 5% carbon dioxide for at least six hours. After the incubation, analyze transfection efficiency with fluorescence microscopy, flow cytometry, or immunoblot.

The most important part of this procedure is to ensure uniform distribution of the transfection mix so that all macrophages come in contact with the same amount of mRNA.