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Help with stable cell lines


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#1 mexemi

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Posted 07 October 2004 - 07:22 AM

Hello everyone. I am trying to establish for the first time a stable cell line. I transfected cells with a plasmid carrying G418 resistance. I have been selecting the cells with G418 for 1 month already. Many cells died but there are still so many cells so I cannot pick isolated colonies with a Gilson. I have read there are two methods to pick cells: either seed them more diluted, let them grow and then pick isolated colonies with a gilson or dilute them to a final concentration of 0.5 cells/ul and seed them in 96-well plates. The cells are a hybrid between mouse spinal motoneurons and neuroblastoma, they seem to grow quite fast. In case I decided to seed them diluted in a 10cm dish, do you have an estimate of how many cells I should seed in order to pick well isolated colonies? I have had a hard time too because some people use always the same dose of antibiotic (selection and further growing) while others use a higher dose for selection and a lower one for growing... what is the best thing to do? Hope anyone can help me finding a solution. Thanx

Edited by mexemi, 07 October 2004 - 07:28 AM.


#2 kant0008

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Posted 11 October 2004 - 09:25 PM

Hi,
as to your amount of selection antibiotic, it doesn't really matter what you use as long as you have healthy cells and enough of a selection pressure at all times. I personally like hitting cells with a high dose, and when only a few are left I decrease the antibiotic.
You say that [QUOTE]Many cells died but there are still so many cells so I cannot pick isolated colonies with a Gilson,

Are you sure that you have stably transfected cells, are you using enough G418? Normally only a few cells survive, so that you should be able to pick colonies.

#3 caro

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Posted 13 October 2004 - 01:53 AM

I think the best method for isolating cells are the one where you dilute the cell suspension to 0.5 cell/100 ul, and then seed 100 ul in each well in the 96 well plate. With this method it is easy to pick up the clones.
Rember to verify that your clone(s) express what you have transfected the cells with, and hopefully that they express it more than the pool of cells you isolated the clone from. This can be done with RT-PCR og Northern blot.
I think the selection method is determined on the basis of what vector you use.

Regards Caro

#4 mexemi

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Posted 14 October 2004 - 05:16 AM

Hi there all. Thanx for your replies. In the end I used both methods and now I am waiting for clones to grow. It should not be a problem screening for positive clones even with a WB since my stable clones are murine cells while the transfecteed gene is human. Both proteins have a slight MW difference that can be discriminated using a 14% PAGE gel. How good is it to lower the amount of antibiotic after you have selected cells? SHould I always keep the same amount? Thanx again!




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