Dear Friends,
I performed a kill-curve experiment and determinated that ; at 400mg/ml g418 consantration only 5-10 non-profilable cell survived at 7 day in 48 well-plate, plated with 50.000 U87 cells. g418 has been added to medium after 24h of seeding. at 600 mg/ml g418 there was no alive cell.
Than I transfected my cells with a resistant gene carrying vector. 24h after transfection I passaged my cells from 12 well plate to 6 well plate. my Transfection efficiency was between %50-%70.
I had 4 conditions Vector with A gene, Vector with B gene, Empty vector as well as non-vector transfected.
at 3rd day massive cell death has been observed in both conditions.
at 6th day whole cells died at non-vector control plate. But the problem is that, other conditions with vector transfecteds are now nearly %80 confluency. But they were at %20-%30 confluency when I passaged them from 12 well plate to 6 well plate.
My problem is that many cells are dividing during g418 selection. Do I need to increase the consantration of g418 ? maybe at first too dense transfected cells degraded g418 so the others has esgaped from selection. Or shall I continue to selection with the same condition.
One more information, When I check GFP expression a few cell (10-30) have very high GFP accumulation, that can be derrived from transfection (1 week before) they may be turned in to stable transfected cells. On the other hands there is a background with lots of cells that have verry verry poorly gfp expression (nearly %40 of the cells) so that can be mean that , the transient transfected cells still have antibiotic resistant so I need to continue selection instead of starting from zero?
Last question ; If the cell dense continiue to increase, can I passage them in to 60 dish from 6 well plate? Or can I split them in to 2 well of a six 6 well plate.
Thanks for answers.
Hi,
I assume you do renew the medium regularly after transfection? It is correct that G418 is metabolized by the large number of transiently transfected cells (at high density) and disappears quite quickly, which I assume is the problem here. You can either increase G418 for the first few days, or which I would recomment renew the medium daily. Remember that even at optimal selection concentrations, non-transfomed cells will require several days to be affected by G418.
On the weak GFP expression in most cells after one week: I would assume that this is residual protein from transient transfection, it takes a few days for cells to completely abolish all protein produced during transient transformation (both GFP and NeoR). It is well possible that the activity from NeoR is sufficient to keep the cells from dying by selection, I would simply keep the selection conditions a bit longer (two weeks) and see if you get a better selection.
I would agree with you that your few strongly fluorescent cells/colonies are stable transformants.
On the cell density, I usually plate my cells direcly to 100mm dishes 48h after transfection for picking clones after colony formation (monoclonal lines). If you intend to create polyclonal lines, it is fine to passage the cells to larger dishes when necessary.
Dear Heinrich,
I am pleasured for your kindly response.
I renewed the medium every 2 days till now. After your advice now I am changing it daily. When I got major cell death at 3rd day I thought that antibody degration is not a such problem. But now I consider about it.
GFP expresion should be derrived from the cells that have vector before proliferation but to day I have checked them again and signal is much lower.
I aim to have polyclonal cells. Now they are nearly %80 confluency. Tomorrow is no major cell death will be observed, I will split them in to 2 diferent well of 6 wells.
Thansk for answers.
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