Dong Chen got up to 70% efficiency, and that's a really good result for a liver cancer cell line like HUH-7. For siRNA, the results are around 80% (https://altogen.com/product/huh-7-transfection-reagent-liver-cancer-cells/) but for plasmids of course the efficiency falls. Given that GFP is a pretty small one to add, it isn't that surprising that the efficiency was so high. However for larger plasmids you'll need to use a complex condenser, and even then you'll still have around 60% efficiency, which is still enough for most research purposes.

Irrespective of your cell passage number, you need to try three different ratios of FuGene 6 and DNA as suggested by the supplier of the FuGene to get best transfection of your plasmid preparation under your cell culture conditions.

Hi Dong

According to my experience with transfection, you should always let the cells to come in log phase of growth before transfection. Not too early and not too late. Usually any cell lines will take 2-3 passage to grow at normal rate after thawing, condition that it must have been frozen very properly (proper DMSO; good quality of cell frozen with counted figures apprx 1X10^6 not more than this). After you have all this in place, you may take up the transfection using any reagent. For Fugene (or any transfecting reagent when using for the first time or any cells for the first time for transfection), always try and titrate the DNA conc. with prescribed transfecting reagent volume in the concerned plate to be used for transfection. For titration, prefer the recommended conc. from the manual of the company and keep a particular ratio below and above what is recommended.

Eg: DNA to transfecting reagent conc.=

1. 5ug+ 1.5 uL (as described commercially)

So, you may choose 2.5 ug DNA and 7.5 ug DNA with reagent (per ug as prescribed).

This will help in better evaluation of transfection journey.

Good Luck!

Agree with both the answers. In addition, you can try your transfection with Lipofectamine. I got good results with this reagent while working with Huh7 cells.

Best of Luck.

When I worked with Huh7s and Fugene HD this was the protocol I used:

Do not pass cells more than 20 times, can start transfecting after 2-3 passes from resuscitating from frozen. Cells were frozen down @ 2x10^6 cells/ml in FBS + 10% DMSO.

I would seed a 24 well plate at a density of 6x10^5 cells/ml and leave the cells to settle for 3-4h prior to transfection. If I wanted to transfect in SFM, I'd change the medium and give it another 1-2h prior to tranfection. I found that if you left the cells to settle overnight, the transfection efficiency reduces - and this could be due to the cell membranes stabilising after trypsination - the membranes of the cell get disturbed when you trypsinate it off the flask surface and this weakens it so makes getting things into the cell a bit easier as it's more leaky.

The limiting step for Fugene HD is the amount of DNA you can physically complex with Fugene - I believe it's 2ug? I transfected my cells at a ratio of 4:1 (8ul Fugene HD to a final vol of 100ul). But as the others above have suggested, it is best to optimise your transfection ratios to get the best efficiency. I normally transfected my cells for 48h then did a wash step and changed to fresh media.

Using Fugene HD, the most I could get was about 15-20% efficiency of transfection - not great for EM work but enough for IF. If you want a higher level of transfection efficiency - have a look at electroporation.

Hope it helps!

Thanks for the above answers.

Actually I ve practice multiple times to have a good transfection protocol and I found that the transfection rate is related to the cell growth rate.

When the cells are in good status, it is easy to have a good efficiency. When I tested it using GFP plasmid, the best could get 60-70%.

However, as John Poh said that, I am not sure whether it is good. Huh7 grows very fast and 6X10e5 may be too much? And the 3-4 hours needed to adhesion to the wall, is it enough?

Now I like to change medium to Opt-MEM and then change it to the complete medium 5-6 hours later. Do I need to wait 2 hours after I change medium as Opi-MEM before transfection?

Wow 60-70% transfection efficiency of Huh7 cells, that's very good!

Again, from what I've noticed throughout the years, Huh7 cells from different research groups vary - some are easy to transfect, some attach well, some grow fast... unfortunately because there is only one source of Huh7 from a biobank in Japan it's harder for all groups to ensure their huh7s are like the original deposit.

You can seed your cells in optimem and transfect straight away, then change to complete medium as described. I used optimem for a quick experiment a while back and didn't really notice any difference in it with my cells.

Dong Chen got up to 70% efficiency, and that's a really good result for a liver cancer cell line like HUH-7. For siRNA, the results are around 80% (https://altogen.com/product/huh-7-transfection-reagent-liver-cancer-cells/) but for plasmids of course the efficiency falls. Given that GFP is a pretty small one to add, it isn't that surprising that the efficiency was so high. However for larger plasmids you'll need to use a complex condenser, and even then you'll still have around 60% efficiency, which is still enough for most research purposes.