Slit cells or add antibiotics, which is first after stable transfection - (Nov/02/2005 )

i am going to do stable transfection. see after you did the transfection, you need to split the cells and add the antibiotics to select. which one first? one collegue said after she did transfection, then split them to 96well then add antibiotic the next day. another collegue said he did transfection, then add antibiotics for couple of days, then split the cells to 96wells continue the selection. I intended the first one. What do you think?

thanks
cathy

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Depending of your transfection efficiency you might have massive cell death (non resistant cells)and therefore splitting the cells afterwards would be maybe more preferable but I guess in terms of efficiency of the selection it won't change too much

pesji

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Hi,

It all depnds on expression of selectable marker. If it takes time to express then add your drug after 1st passage. If expression is immediate then add your drug prior to split. I hope this helps.

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aha...
depends on the mechanism of your drug. I think that easy drugs are effective whithout a split.
But in most cases drugs need active cells for acting. So you are supposed to split cells and add drug.
You can do both in one step (resuspend cells in medium supplemented by drug)

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i am going to use Zeocin. what do you think of this one

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The concentration of Zeocin required for selection of transformants is pH and salt concentration dependent. The higher the pH and the lower the salt concentration, the better is Zeocin activity.
Selection of common E. coli strains is achieved at 25 µg/ml zeocin in Low Salt LB Agar medium adjusted to pH 7.5 .
Selection of animal cells: 100 to 750 µg/ml depending on the cell line.
Selection of plant cells: 50 to 250 µg/ml depending on species and media.
Selection of yeasts: Depends on species and media. S. cerevisiae Sh ble transformants are selected at 50 µg/ml in YEPD at pH 7.0 .

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bleocin family drugs (which belongs zeocin) perturb plasma membranes (supposed) but their activity is generally believed to be correlated with their ability to bind DNA by intercalation. The DNA is degraded by the metal ion chelating portion of the molecule which forms an active complex with iron II and molecular oxygen.
So i assume it's a active-cells-selecting antibiotic.
So split cells and add drug.
I've succesfully tested it on the Hek293 cells and got clones in about two weeks.

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I just did a killing curve on my cells, 100ug/ml of zeocin kills almost all cells. so I think I will first split, then add the drugs. just more work to do than add first, split

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ok good choice and good luck

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I just did the transfection. the cell I am working is suspension cells, so I need to split into 0.5cells/well. So first split, then add antibiotics. so lots of cells are negative ones, I might end up with very few colonies with paying so much work. So I think might transfer to 6 well plates and select for a weekend, then split as 0.5 cell per well. what do you think

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