after 6 weeks of selection in G418? - (Feb/08/2007 )

Dear All
I have prepared stable transfectants using pCDNA vector, performed selection for 6 weeks in G418 and picked up isolated colonies. the strange thing I am having now is that clones with empty vector and those with vector harboring my insert are resistant to induction of apoptosis by my treatment as compaered to parental cell line. I mean the effect of my treatment is masked by that abnormally imparted resistance to both types of clones irrespective of the insert.
I do not know what is the reason of such resistance and how to overcome ? should I , for example, seed the cells in medium free from G418 before addition of my treatment?
I appreciate your comments
thanks

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What are you using to induce apoptosis? Try to use different concentration of the chemical to induce apoptosis.

Also I have not added G418 when doing the actual experiment.

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well, on parental cell line my combination treatment induces about 70% apoptosis in many replicate experiments. When I applied this combination treatment to both stable transfectants(empty vector & vector with insert) surprisingly that percentage dropped to 5% in both types of clones. I did a positive control (using parental cell line) and the percentage of apoptotic cells was about 70%, so definitely it is not a matter of experimental error. the question now is when should I withdraw G418? is it 24 hrs before addition of my combination treatment or should it be withdrawn earlier?
thanks scolix

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I withdraw G418 when I plate the cells in the culture dishes. I dont think it should matter if you withdraw G418 24 hrs before the experiment.

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unfortunately withdrawal of G418 did not affect that induced resistance to apoptosis.
I do not know why both types of clones (empty pCDNA vector , and pCDNA-INSERT vector) became resistant to induction of apoptosis?
any comments????????????????????????????

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