Optimize Lipofectamine and DNA ratio, and transfection time. If you use SFM during transfection, cells may die. You can use 1 - 5% serum containing medium.

Hi Mohammed,

It looks that you need some optimization!

As Chandra said, Lipofectamine/DNA ratio, incubation time and the medium are all important things. But also the cell density, here you give a number of 10.000 cells, I assume it's in 96-well... you should determine the right density empirically.

And you should consider whether you have selected the good reagent. Lipofectamine 2000 is a very versatile reagent but not the most performant. In the family, Lipofectamine 3000 could be better. But keep in mind that Lipofectamines are first very expensive, and that you have plenty of other reagents available on the market. In addition, lipid-based reagents are known to induce toxicity or physiological changes to the cells.

For some optimization, please take a look to the attached manual of the Viromer RED, our reagent for plasmid transfection. From page 9, you will find tips for optimizing the amount of transfection complex, the reagent/DNA ratio, the time of incubation or the cell density. It works for any other reagent!

If you want to try another chemistry than Lipofectamine, you can start with the Viromer RED, it's a polymer-based nanoparticulate reagent featuring a viral mechanism of membrane fusion, resulting as main effect into an active escape from the endosome. Please, let me know, I can organize a free sample for you, simply write me at [email protected]

Best,

Sandra

Hi Mohammed Ahmed

I had similar problems transfecting some (adherent) cell lines with lipofectamine 2000 and LTX.

I think you could consider some suggestions:

1.- Given that the efficiency of tranfection for your cells is low; you could increase the number of cells to be transfected (increasing proportionally the quantity of DNA) in order to get bigger number of positive (GFP) cells.

2.- Confluency is important for growing cells too, specially after toxic stimulus (as you correctly said about the effect of lipofectamine); you could allow the cells to grow for longer time before transfer them to bigger flasks. You could also increase the percentage of serum in your medium for your treated cells (for example, 5% to 10% or 10% to 15%).

3.- You could measure again the quantity of DNA in your stock and how much you are using and comparing with other transfection protocols. For example:

https://www.thermofisher.com/mx/es/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-raw267-3-macrophage-cells-using-lipofectamine-ltx-reagent.html#1

Hope it helps!

Regards.