Hi all !
I need a smart sugestion for the G418 selection that I need to do for my cell(purpose to make stable cell lines expressing my inserts, cloned in pc1-neo)

1)I decided the dose of G418 that need to be used for my cells(I USE K562 human erythroleukemia cells)

2)For how long do I need to challenge the drug, what I mean how often, say I challenge G418 , 48 hrs after the trasfection, a] then when NEXT do I need to challenge,[/b]after every 24 hrs, or 48 hrs or when??

3) Is the G418 dose keep constant throughout the entire selection process or do I vary it, slowly bringing it down/or /elevating it??

4)G418 seems to be quite acidic as the pH of the media alters a bit(though we add a little, still), so whats tbe best vehicle which can be used to deliver it:

a) just aqeous /water
in some buffer: IF YES then
i) PBS
ii) HEPES


Thanks a lot for valuable suggestions

waiting for the response

Subhradip

Hello Subhradip!

I usually trypsinize my cells two days after the transfection and put the cells of one plate onto four new ones.
The next day I start to give medium containing the G418 dose I decided to use. You have to keep the high concentration of G418 until your untransfected control cells are dead (it normally takes about one to two weeks). Then you can bring the concentration of G418 down - but to keep your clones stable you have to feed them with G418 containing medium all the time. The medium you change as often as you do with your untransfected cells.

In our lab we solve the G418 in PBS.


Greetings,
Chakchel

You need to establish a killing dosage curve first for non-transfected cells first. Once you know the lowest dosage that kills 100%, use that for selection.

Do it 24 to 48 hr post transfection. Low cell density helps elimination of non-resisting cells.

You can use 1/2 the dosage to maintain selected cell line once it is established.

Acidity is due to it is a salt form with some leftover acid. You can bring the pH back with NaOH.

Thanks Chakchel
That's a wonderful suggestion that sounds perfect to me, I will follow this and post you further in case I get some roadblock(not hope so !!)

Thanks again
Subhradip

You are welcome.
Good luck!

You can solve G418 in 100mM HEPES, and bring pH to 7.3 with NaOH.
Before your selection experiment, you should do a killing curve assay in your pre-transfected cells.
Good luck.

Andy Kwong