I have transfected GFP-tag plasmid to THP-1 cells, and hope to check the efficiency. I used 24-well and there were 5*10^5 cells per well. I used 50ul opti-MEM mixed with 1ul lipo, and used another 50ul opti-MEM mixed with my plasmid.
24 hours later, I checked the cells by fluorescence microscope. There were approximately 20 cells with fluorescence each well. The efficiency was about 1 to 10000.
Although the suspension cells have a low efficiency in transfection, but I am wondering if it is normal or not? If not, I have to check my technology of transfection.
1. Cell number looks to be high for 24-wells.
2. Amount of lipo looks to be low for this volume (a few one of transfection reagents can work at that range at my best, and that is their lower limit).
3. Dna amount (?) and quality is important.
(human) macrophages is hard to transfect, anyway. Try to modify your protocol. Good luck.
Important thing - check whether your DNA is contaminated with LPS or not? For this, just treat your cells with your DNA alone (without reagent) and check TNF-a or other established marker by PCR. If your DNA is contaminated, you should not use the DNA, even if you succeed in transfection. Then, try seeding cells on serum free and antibiotics free condition before transecting. Use only low passage cells. Try different transfection reagent at different DNA ratios. Good Luck.
I got similar results with some conditions of liposome. Electroporation worked much better. There is a recomennded protocol from manufacturer for THP-1 cells.
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