Toxicity is commonly observed with Lipfectamine 2000. We also found considerable differences between batches both in toxicity and also transfection efficiency. If a batch is not acceptable we ask the provider for a replacement.

We typically use various cell lines. Hela cells, for example, seem quite sensitive (again, this depends on the batch). To solve this one can titrate the amount of Lipofectamine used (keeping the ratio Lipo/DNA constant), limit the exposure (incubate cells only for a couple of hours or so, then wash and replace with fresh medium), and determine the optimal cell density. Higher cell density (e.g. 80-90%) minimizes toxicity-induced cell death. It also makes a difference whether cells are attached to plastic or a cover glass during transfection, plastic being preferred. Finally avoid splitting cells right after transfection and let them recover o/n. In any case, even if cells survive, expect a delay in cell cycle progression.

Toxicity is commonly observed with Lipfectamine 2000. We also found considerable differences between batches both in toxicity and also transfection efficiency. If a batch is not acceptable we ask the provider for a replacement.

We typically use various cell lines. Hela cells, for example, seem quite sensitive (again, this depends on the batch). To solve this one can titrate the amount of Lipofectamine used (keeping the ratio Lipo/DNA constant), limit the exposure (incubate cells only for a couple of hours or so, then wash and replace with fresh medium), and determine the optimal cell density. Higher cell density (e.g. 80-90%) minimizes toxicity-induced cell death. It also makes a difference whether cells are attached to plastic or a cover glass during transfection, plastic being preferred. Finally avoid splitting cells right after transfection and let them recover o/n. In any case, even if cells survive, expect a delay in cell cycle progression.

Thanks. This is really great. I never realized that there was this significant difference in batches. Do you also go for 2 hr transfections. I always did the recommended minimum of 4 hours. Great sugestions.

2 h transfection works, but might reduce transfection efficiency, it all depends on your needs. In my experience cell density is a major factor as well as presence/absence of additional stresses (coated glass as substrate, splitting, drug treatments,...).

I've also observed toxicity with lipofectamine 2000 (as well as LTX but lower). Transfecting at 80-90 percent of cell density by 4h and then change of the transfection medium solved the problem for endothelial and melanoma cells.

Besides you could lower the amount of DNA/lipo complexes, using 100-200ng complexed with 0,6ul lipo in 100ul medium by 1h before add to cells in p35 dish with 900ul medium, obtaining 50-80% of transfected cells.

Have you tried mixing plasmid with lipofectamine 2000 first then pour the cells on top of it? I have tried a few times and seems to work for my primary culture endothelial cells.

I have found that in high concentrations, lipofectamine is toxic to HeLa cells, and as previously mentioned, the cell number seeded has an impact on this.

While the manual may suggest a 1:3 ratio of DNA to Lipofectamine reagent, I have found that minimizing the lipofectamine concentration as much as possible reduces cell death. I have good results from using DNA to Lipofectamine ratio of 1:1 and 1:2.

1)Did any one does know what is the pDNA to lipofectamine2000 (invitrogen: 11668-027) is preferable with LNCaP cells, to get high tranfection efficiency?

2) Transfection after ~one day of seeding or tranfection along with trypsinized cells: which one gives better result?

3)What is the reason behind the 5min incubation(before mixing DNA& Lipfectamine2000)?

4)Do using the eppendorf/plastic plates has any effect with lipofectamine reagent etc. during transfection?

 Did anyone have experiences use lipofectamine 2000 in long culture primary neuron?

Hi guys, I have been reading the comments above. I have been trying to express GFP in HeLa and HEK but have obtained no results on ECL film. I have used lipofectamine 2000. is there a specific L2000 to nucleic acid dilution ratio at which transfection can be enhanced? does anyone know any research papers where they have mentioned the failures doing anything similar? I have a submission in 8 days and need to know what all can go wrong. any suggestions please?

Has anyone experienced very low transfection efficiency using Lipofectamine 2000? - ResearchGate. Available from: https://www.researchgate.net/post/Has_anyone_experienced_very_low_transfection_efficiency_using_Lipofectamine_2000#56ee804e217e20dad4160676 [accessed Mar 20, 2016].

Hi Vishwesh 

Did you find any research papers about the topic? 

Toxicity is usually common with generic reagents being used in very unique cell lines that they weren't exactly designed for. There's literally hundreds (https://altogen.com/products-index/) and depending on the cell line there can be different optimization protocols and such. Following pre-optimized protocols is a good way to ensure successful transfection, and you should definitely consult the literature out there for any particular tips and tricks.