Hi,

HepG2 are not happy when alone. If single clone is needed, try to grow them in a mixture of fresh and used medium. Grow untransfected HepG2 in a separate dish - without ATB, let it grow for couple of hours, take the medium (growht factors ... enriched, without cells) and mix it with fresh medium 1:1, add ATB and use for your transfected cells.

Alternatively, you can markedly increase % of serum - lonely HepG2 should survive and start dividing.

If everything fails, you can try another methods of getting single clone (cloning cylinders, cover glass method, etc).

If your ATB selection works perfectly, you can add untransfected cells to one transfected cell, grow them in ATB free medium together, until they start growing. Then kill the untransfected with ATB. A bit tricky, I would prefer methods above.

Martin

Hello Eriks,

What is your transgene doing? You overexpress or silence a gene with a vector or short hairpin siRNA plasmid?

When the selected cells die it can be reason that the transgene affects them so severly that they cannot survive. Then you have to equip your plasmid with a tetOn or tetOff promoter to activate transcription by adding or removing tetracycline (On or Off) when you want to have the effect and your stable cell line is normally growing.

Several cell lines are known to need neighbours to grow, if there are no other cells they turn senescent and die!! In this case you used your selected cells and seed a single cell into ten 96 well plates and hope that a few of these 1000 cells start growing. Would be worth a try when you have selected cells!

Have a look on this chat there are more suggests:

https://www.researchgate.net/post/I_tried_transfecting_uch1_cells_with_Lipofectamine_2000_but_very_low_percentage_of_cells_were_transfected_successfully_Is_there_a_better_alternative

Regards

Hi,

HepG2 are not happy when alone. If single clone is needed, try to grow them in a mixture of fresh and used medium. Grow untransfected HepG2 in a separate dish - without ATB, let it grow for couple of hours, take the medium (growht factors ... enriched, without cells) and mix it with fresh medium 1:1, add ATB and use for your transfected cells.

Alternatively, you can markedly increase % of serum - lonely HepG2 should survive and start dividing.

If everything fails, you can try another methods of getting single clone (cloning cylinders, cover glass method, etc).

If your ATB selection works perfectly, you can add untransfected cells to one transfected cell, grow them in ATB free medium together, until they start growing. Then kill the untransfected with ATB. A bit tricky, I would prefer methods above.

Martin

Volker,

I try to make stable overexpression with different plasmids. Works fine with HUH7. Don`t think protein is toxic or something like this.

Martin,

Yes, I think this is a case - they don`t like to be alone. I will try the trick with medium and use of cloning cylinders.

Thanks a lot.

E.

As we did, the hepG2 cell is density dependent, we can't culture it from one cell in our lab until now

HepG2 cells like to be in clumps. In culture situations, harsh passage at very low density, it will take very long time for the cells to catch up, in most cases, single cell in any isolated micro dish tends to die,

There are two ways you can get around, as I stably established a BAC transgene in this cell line.

You can dilute cells to certain concentrations, such as 10^3 -10^4 cells /ml in good suspension, and plate these cells in 6 well plate or bigger dish, wait for week or two for the colonies to appear. Then isolate the single colonies under microscope, and transfer into new culture plate at appropriate volume to expand.

Second method is to dilute the cells to 10-100 cells per 200 ul, and plate them on to 96-well plate, you may need to have 6 plus few plates as the efficiency of single colony is not going to be high. Keep monitoring for two to three weeks for the colonies to appear, then you can screen for the well(s) for single colony, subsequently expand the colony into bigger volume.

Best of Luck

Lingli

Thanks Lingli. Sounds good.

My experience with sorting HepG2 cells as single cells into individual wells of 96-well plates is that most did not survive. Sorting at a higher number per well (5 or 10 cells per well) did produce more survivors. But, since these survivors all but twice appeared to be individual colonies once they grew, I believe the "greater" survival rate was only due to having 5 or 10 chances at a surviving cell per well rather than anything other reason.

I am currently planning on altering my protocol to sort the cells directly into conditioned medium in hopes that it will provide sufficient 'survival' signals to increase my yield. I did eventually use conditioned media last time, but it was downstream to try to promote growth of the colonies that did result. I didn't really notice much growth improvement, but it may have been too late by then.