2 replies to this topic

[bkool4ya]

Hey guys,

I am doing co-transfection with 293 cells with home-made 'lipofectamine' like protocol.
So I seed them on Day 1, Transfect on Day 2, change the media on Day 3, and lyse them on Day 4.
But after transfection on Day 2, the cells seem to be less viable and Day 3, I don't see them growing compared to just control plate with not transfection.
I seed 750~900000 cells per 10cm dish.... I don't know what the problem is...can somebody help ?

[bob1]

At a guess it would be your transfection reagent (let me guess... polyethyleneimine?), but it could also be that your construct is toxic, you have too few (or too many) cells, absence of serum for 24 hours(?)...   Do you have a transfection reagent only control?

Many cells do not do well with transfection as it is, you could try optomising the amount of transfection reagent and/or DNA you are adding.

[Elle]

I agree its most likely either the number of cells you are seeding, the concentration of your transfection reagent or the amount of DNA you are adding. I do Lipofecatmine transfections in 6 well plates and generally get good efficiency and less cell death if I seed 250 000-500 000 cells per well of a 6-well plate (9 cm I think) on day 1, then transfect on day 2 then harvest on day 3 or 4-I generally dont bother washing as I find I just lose more cells that way. I use 4 ul of Lipofectamine and 3 ug DNA per well and it works like a charm. You should also try running 2 controls: a control thats just cells in the half serum media and another control that has cells+transfection reagent+half serum media. Hope this helps