I use Lipofectamine 3000 to deliver plasmid DNA into Hep G2 cells, frequently. The transfection efficiency of Lipo 3000 is higher than that of Lipo 2000. I use 7.5X10^5 cells/well in 6-well plates and 2.5 ug DNA/well. I have tried reverse and the traditional transfection protocols. Reverse transfection will lead to much higher cytotoxicity than traditional one. I usually obtain >60% transfected cells.

I use Lipofectamine 3000 to deliver plasmid DNA into Hep G2 cells, frequently. The transfection efficiency of Lipo 3000 is higher than that of Lipo 2000. I use 7.5X10^5 cells/well in 6-well plates and 2.5 ug DNA/well. I have tried reverse and the traditional transfection protocols. Reverse transfection will lead to much higher cytotoxicity than traditional one. I usually obtain >60% transfected cells.

Thank you Shang-Hsin Wu for your answer but I want to clarify about something. Did you use 750000 cells/ well in the reverse or forward transfection? and what was the concentration of lipofectamine you used ?

HI, Razan Masad,

In the beginning, I used the same cell number (750000 cells/well) to apply the forward and the reverse transfection parallel. The DNA amount is followed the instruction of Lipofectamine 3000 (2.5 micr-gram per well of 6-well plates). I have tested the ratio of DNA: Lipo3000 from 1: 0.5, 1: 1, 1: 1.5, and 1: 2. The ratio of 1: 2 obtained the best result but the cytotoxicity and the cost were higher. So I now use DNA: Lipo3000 = 1 ug: 1.5 uL. (The ratio of Lipofectamine 3000 to P3000 is 1: 1. Do not change the ratio because the transfection efficiency will be reduced dramatically.)

I think that the cytotoxicity is majorly come from the reagent P3000. I have used Lipofectamine 3000 without P3000 to deliver siRNA by reverse transfection, which is also applicable and merely no cytotoxic. But the amount of siRNA will be required more.

Hope these information to be benefit to your studies.

The efficiency of transfection HepG2 cells by using Lipofectamine 3000 transfection reagent can be reached 70-80% efficiency when we used EmGFP plasmid (size of vector ~6Kb). There are two important steps:

1> Plate right density of cells:

HepG2 cells tend to grow in clusters and top to each other, not easy to make them as single cells when you split them before plating in culture plates or subculture in flask. You need use 10ml pipette with 200ul tip on the head of pipette to up and down 5 times after you trypsin your cells and add 10ml growth medium. Based on my experience, trypsinize HepG2 need 10mins or even long time in 37C incubator after putting TrypLE solution. Once you get single cells you plate them  in culture plate which you prefer. For 6-well plate, we seed 700000 cells/well (the cell number is depended on how you count the cells), but at the next day, the cell confluency should be around 65-75%.

2> Transfect the cells:

The next day, transfect the cells as following  for per-well of 6-well plate:

a) Prepare two 1.5ml eppendorf tubes labeled as A and B, add 250ul of Opti-MEM I in each tube

Tube-A + 6ul of Lipofectamine 3000 reagent, mix well

Tube-B + 2.5ug of DNA (your plasmids) + 5ul of P3000 (DNA : P3000 = 1ug:2ul),  mix well

b) Combine Tube-A and Tube-B by adding Tube-B diluted DNA/P3000 to Tube-A diluted transfection reagent, total volume ~500ul.

c) Incubate the combined solution at room temperature for 10mins

b) After 10mins incubation, add the DNA/P3000/tfx reagent complex mixture to cells

d) Post-tfx 48hrs, harvest cells and perform analysis assay. If your report gene is luciferase, you may harvest cells at post-tfx 24hrs.

Reverse transfection works, but efficiency will be lower than forward one. The cell number is 2.5x of forward transfection.

Keep in mind, HepG2 culture is very important for your experiments, always make them as single cells when you passage them.

Hope this will help you.

Thank you Shang and David ! That was so helpful 

Hi David. I've also been trying to transfect HepG2 cells, and the transfection efficiency has really been poor. I'm thinking it might improve if I follow your advice, however, from my experience, leaving the cells too long in the incubator (with trypsin/EDTA) damages them. The average time I leave them in trypsin/EDTA is 4 minutes. I've tried 5 to 7 minutes before, but they didn't look very healthy the next day. Could you please advise me on this. I strongly think the transfection efficiency would improve if I follow the steps you outlined above. 

Thanks a lot,

Bright 

Hi, Bright Ozuruoke Enyindah,

I use Hep G2 to perform my experiments a lot. In my experiments, it is safe to detach Hep G2 cells by trypsin/EDTA for 7-10 minutes at 37C. From your description, I will suggest you to do some assays:

1. Mycoplasma contamination. 

Mycoplasma can secret nuclease which is damage to exogenous plasmids.

1.1 You could use PCR to amplify the mycoplasma-specific genes to determine the contamination. Some commercial kits are able to do this.

1.2 You could take some plasmid DNA incubated with the culture medium at 37C for 1 hour, then perform electrophoresis. If the signal of plasmid DNA becomes smeary or lost, your cells are contaminated.

1.3 Take some cells to stain by DAPI. If the single of nuclear becomes strange, give away those cells because they are not healthy.

2. To determine the suitable cell density.

The morphology, the replication time, and the "health" of hepatic cell lines are influenced by cell density. I plated Hep G2 with different cell densities then observed in 24, 48, and 72 hours to obtain the suitable cell density for my further assay. Now, I always passage my Hep G2 cells every two days. 

3. Replace the new Hep G2 cells. 

4. The ratio of DNA/Lipofectamine 3000. I have tested the ratio to 1: 2 as the suggestion from David. The results were good; but the cost and cytotoxicity were high, especially to those exp. required to incubate longer than 48 hours after transfection. 

Good luck,

Shang-Hsin

Transfection of HepG2 cells can occur at up to 90% efficiency if you're using siRNA and appropriate transfection reagents (https://altogen.com/product/hepg2-transfection-reagent-hepatocellular-carcinoma/). HepG2 is known for being resistant to transfection, and having gentle protocols will help you in the long run. I agree with all of the points made by Shang-Hsin, and I'll also add that monitoring the expression at consistent time intervals can help you determine if there are post-transfection factors affecting your cell culture.

Shang-Hsin Wu : Hi! Can you tel me whether lipofectamine 3000 requires medium replacement after 6 hours. And If you need to treat the cell with drugs what would be the best method? Is it better to remove the medium and add drug with culture medium or to add the drug prepared in culture medium (Conc calculated accordingly) to the lipofectemine containing medium

Vini Ravindran :

I DONOT replace fresh medium after transfection 6 hours and the cell lines that I used are Hep G2, Huh-7, Mahlavu, HK293T and HeLa cells. However, you may do that for your cells if your cells are sensitive to transfection reagents. In my experiments, I need the protein exogenously expressed and then treat transfected cells with drugs. The shortest time point that I could detect the tagged protein (FLAG and HA tags) is 6 hours after transfection in Hep G2 cells. I will suggest you doing a pilot study for your targets.

For drug supplement, I will prepare fresh medium containing those drugs what I need to replace the medium containing transfection reagents. I will add drug-containing medium via pipet-man to minimize the variation between wells. Adding drug directly into each well may increase the variation of drug effect among wells.

it's a old question but let me give some new input. We, at Lipocalyx launched recently a new reagent for plasmid transfection because our Viromer RED showed some limitations and HepG2 were one of the issues. See on the graph how we improved that: it's a forward transfection of a luciferase encoding plasmid of 3.5kb using 100ng DNA/96-well (n=4) folowing manufacturer's instructions. We did not count transfected cells but the global protein expression.

> the product is named VIROMER PLASMID.