Transfection efficiency depends on several factors; the size of your plasmids, the transfection reagent, and the competency of the cells. HepG2 isn't really that difficult to transfect with siRNA (with efficiencies up to 80% with this kit https://altogen.com/product/hepg2-transfection-reagent-hepatocellular-carcinoma/) but for larger plasmids you'll need to condense them down in order to have them enter the cells.

Hi,

in our hands, the crucial factor influencing transfection efficacy in HepG2 is initial confluence of cells. At 50% confluency, we are observing 70-90% transfectant. When the confluency is higher, efficacy drops significantly (eg at 70% confluency, we observe less then 30% transfectants). We use LTX lipofectamine.

Thank you Martin. That's good to know. I always use very confluent cells, >90%. I tried to lower confluency before. However, when I used about 70% confluent cells, they did not grow faster and the density became very low after 4 days post transfection. 

When you use 50% confluency for transfection, what medium do you use to grow cells? do you use DMEM/high glucose supplemented with 10% FBS? If the cells are dividing actively, will they lose the plasmid? If you get about 100% cells transfected at 50% confluency, when the cells become confluent say at day 3 post transfection, would you expect only 50% of the cells are expressing your protein? 

One concern for me is that I don't wanna cells actively divide after transfection. I suspect that it may cause the loss of intracellular plasmids. I am not sure though. 

Thank you

Hi Masarrat,

Thank you for your reply. I wonder 4 day post transfection, what's the percentage of cells are expressing the protein generally

 To address your question about endotoxin removal, you can use the Triton X-114 phase separation method.  It work quite well, but there are two things to be aware of.  First, your post-purification plasmid solution will have some Triton in it, which could interfere with your liposome-based transfection.  Second, you will lose a little bit of the plasmid during the process.

The Triton X-114 method (as well as other methods of LPS removal) are nicely reviewed in an article available on Research Gate:

https://www.researchgate.net/publication/6112200_Methods_of_endotoxin_removal_from_biological_preparations_a_review

Article Methods of endotoxin removal from biological preparations: A review

Hi,

Dear Tan. How about your work on Hep G2 cell transfection. I tried Hep G2 transfection last week using lipofectamine 3000, and the initial confluency is about 70%. However, the efficiency is very low , less than 20% even 72hs after transfection. 

Transfection efficiency depends on several factors; the size of your plasmids, the transfection reagent, and the competency of the cells. HepG2 isn't really that difficult to transfect with siRNA (with efficiencies up to 80% with this kit https://altogen.com/product/hepg2-transfection-reagent-hepatocellular-carcinoma/) but for larger plasmids you'll need to condense them down in order to have them enter the cells.