Does anybody know a resource for the concentrations of different antibiotics (like Hygromycin, Blasticidin, G418 etc) which should be used for the generation of stable clones in different cell lines?
Of course one could perform a survival concentration-dependent curve on his own, but I am sure for a "common" cell line like NIH3T3, HepG2, MCF7, etc. this data is already available. Would be great to have a reference with comprehensive collection.
Maria,
This is an excellent question, but to my knowledge I am unaware of any database that exists with this information. The reason for this is most likely because culturing conditions (e.g. media, supplements, humidity, temperature, etc) vary dramatically from lab to lab, which would change the susceptibility of the cells to the respective selection agent.
In our laboratory, we perform a kill curve on every stable cell line we generate, even if it is the same cell type we have used before. We have found that depending on the passage, lot, freeze down, etc. the selection conditions change. If you are in a hurry, you can always choose a "median dose," but this may not always work. Below are our typical ranges of selection:
Neomycin (G418): 100-1500 ug/mL
Hygromycin: 10-1000 ug/mL
Puromycin: 1-10 ug/mL
Blasticidin: 2-20 ug/mL
I hope this is helpful, and good luck with your experiments.
~Adam
Thank you very much, Adam, this is already very helpful. I wonder that you really observe such a dramatic changes in selection conditions upon culturing. Well, though I can indeed imagine that probably the older passages are more sensitive towards selelction. Maybe we can start here a list of tested cell lines for which one already did a "killing curve" just to give an impression of a "reference point"...
IHH (immortalized human hepatocytes) (ca. P7) - 500 µg/ml of Hygromycin
HuH7 (human hepatoma cell line) (ca. P11) - 500 µg/ml of Hygromycin
HepG2 (human hepatoma cell line) (ca. P10) - 400 µg/ml of Hygromycin
HepG2 (human hepatoma cell line) (ca. P10) - 2 µg/ml of Blasticidin
I actually need G418 range for hepatoma cell lines and just to lazy and to impatient to wait another two weeks for a result. But it´s probably unavoidable...;)
it is important to use the same conditions for your kill curve as for your transfected cells, because the success of your selection will depend on plating the correct number of cells.
So other researchers concentrations might simply not work.
One other reason to do kill curve is that some cell lines are insensitive to some antibiotics, G418 being a typical case.
You should also pay attention at the potency of your antibiotic if use powdered form that you prepare your-self, instead of the more expensive ready made solution.
Dear Maria,
pls refer to the web link below,
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Transfection/Selection.html
Though it gives you an idea about the range of selection drugs to be used for prokaryotic and eukaryotic organisms,as previous posts suggests it is better to do a kill curve for your cell line.
I've observed too, there is a variation within the same cell type depending on the cell passage, lot, seeding density etc
so, the bottom line is, as you said, it's probably unavoidable :)
good luck!
chaya
The best way out is to do survival curve. I have different people in the same lab come up with different concentrations. So bottomline, the right concentration is unique for each reseracher.
Thanks a lot for your comments - now I am confident, I have to do it on my own ;)
Best wishes from Stuttgart,
Maria
I agree with all previous comments. We have established stable clones from a CHO cell line that was sensitive in our hands to 25 µg/mL of G418 while most people in the literature begin selection at 100µg/mL. We began our screening at 25 µg/mL and the clones analyzed at this step expressed about 1µg/mL of the antigen. We then increased the concentration to 50 µg/mL and we obtained a clone expressing about 300 µg/mL. It contained 12-20 integrated copies of the plasmid , and was stable both at genomic and expression level for at least 30 passages. The risk with beginning a selection with a too high concentration is to generate clones with hundredth copies that will not be stable after few amplification passages
Some of the info is listed on Life tech web site, I hope it helps you.
http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/transfection/selection/puromycin.html
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