Hello all,
One of my supervisors told me that when transfecting HEK293FT cells, the P3000 reagent is not really necessary. But in other thermo fisher protocols, it says to use the P3000 reagent with the lipofectamine 3000. This got me wondering, what is the P3000 reagent? What additional perks, if you will, does it have in transfections?
Dear Andrew,
when using HEK cells we did not see a big difference if using P3000 or not (it was very effective in both situations). Anyway, you can try it without, but I don’t see any reason to exclude it.
I do not know what P3000 is exactly (may you can find something in the patent, but probably this figure will answer some of your questions:
https://theory.labster.com/mechanism-lipofectamine-3000/
This serves as a buffer for the dissolving plasmids. The non-essential ones can be replaced by the DMEM
Carsten Geiß thanks for the information! And the figure! The figure really helped!
Luwen Zhang ohh I see, so P3000 is like ammonium bicarbonate or tris buffer but for transfections? usually buffers are for matching the pH, does this mean that P3000 also serves to match the pH intracellularly?
Hello Andrew Kim
To transfect cells with double stranded siRNA, it is suggested not to add P3000 reagent. However, addition of P3000 component is recommended for DNA samples. The single use of Lipofectamine 3000 represents a great advantage over the combinational use of Lipofectamine 3000 and P3000 by reduced toxicity. The addition of P3000 contributes to an increase in cytotoxicity of Lipofectamine 3000.
The benefit of adding P3000 reagent will depend on the cell type. In some cells the addition of P3000 may not show any difference in transfection efficacy. However, reduced transfection efficacy may be observed in some other cell types if P3000 is omitted.
So, I suggest you listen to your supervisor as P3000 reagent may not really be necessary when transfecting HEK293FT cells.
Good Luck!
I have no definitive answer to what P3000 is. But if I had to bet on it I'd say it's some version of PEI that complexes with the DNA and improves loading of the liposome. If you have a strongly expressed plasmid and a high concentration it probably doesn't add any advantage. Especially with 293s which transfect so well using lipofectamine 3000 is almost a waste
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