I was trying to transfect HL-60 cells with plasmid containing AcGFP using Lipofectin in Opti-mem. But every time I failed. The cells were stained with DAPI. When observing under fluorescence microscope I only found DAPI signal but no GFP signal. I used 24 well-plate, 1x10^5 cells/well in RPMI 1640 only; plasmid 1 ug, and lipofectin 2 ul in 30 ul opti-mem for 12, 24, and 48 h. What would be the possible reason for the failure? I tried several times altering the lipofectin and plasmid concentration.
Hello,
HL-60 suspension cells are hard to transfect with lipid-based reagents. As you have really no GFP signal at all, it's not relevant to suggest you trying some optimization of the cell density or reagent:DNA ratio... you can have a look to the link below, it's an example of a very straigforward optimization for another reagent (Fugene HD)... maybe good for you. If you want to test an additional reagent, I can provide a test sample of Viromer RED, do not hesitate to ask me. Bests, Sandra.
http://www.analytica-world.com/en/whitepapers/67146/optimization-of-transfection-conditions-for-the-human-hl-60-promyelocytic-leukemic-t-cell-line-using-fugene-r-hd-transfection-reagent.html
Hi,
I also had no luck with lipofectamine transfection of HL60 cells, as they are "hard-to-transfect" suspension cells. You may want to try electroporation. I had some success with Amaxa nucleofector, the downside is that electroporation-based methods will result in high cell death. Unfortunately, Amaxa kits are quite pricey, but if someone has the Amaxa device in your institution, I'd recommend giving it a shot. Best of luck!
Hi,
The question of transfecting HL60 cells seems to be a reoccuring theme, for both DNA and RNA. I agree electroporation is likely to be the best method, however if you do not have access to an electroporator, you could try X-tremeGENE HP DNA Transfection Reagent from Roche, I know several have used this very sucessfully. Good luck!
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