Stable transfection in suspension cells

Kamalian, Laleh via methods%40net.bio.net (by L.Kamalian from liverpool.ac.uk)
Fri Jan 18 17:49:02 EST 2008


Hi to everyone.  I am a PhD student and new to this mail list.  I have a problem I would like to ask.  I am working with cells which grow in suspension.  I have managed to get a good nock down in transient transfection using a plasmid vector and Lipofectamine as the transfection reagent.  Now I am ready to perform the stable transfection.  However I haven't got a clue how I will be able to separate the dead cells from the live ones in the selection process especially after the massive death happens.  Furthermore, I don't know how I'm supposed to pick a single clone even if I manage to get the live ones.  No one in our lab has ever transfected cells in suspension.  The selection method I will use is antibody selection (G418) for which I have already started the optimization experiment in order to obtain the killing curve.  I was wondering if any of you have worked with suspension cells. Could you please give me some help?  Thanks  
Laleh.


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