I did transfection for CHO cells
1- the positive control complete dead (G814 on un-transfected cell)
2-the transfected cells survive but in flow cytometry show me that there are many cells are un-transfected) how ?
This shows the selection is not complete. I Don't know how you confirm with flow cytometry ?;In my experience
1.The antibiotic addition time and concentration are important.
2.Transfection may be failurebyinsufficient DNA, high cell concentration and handling error.
In these conditions, all cells grow around (20%) and after 8 or 9 days all cells start dying and you are not able to get colonies.
I usually allow for 2 weeks of selection, so if you haven't done so already, you might want to analyze the cells then. How certain are you that the plasmid you are transfecting with his 'clean', ie. not contaminated by another plasmid that is empty vector, or by a vector carrying a gene-for-flow-analysis inactivating mutation? If you looked into both of these things, you might consider increasing your antibiotic concentration; since you're analyzing by flow, is sorting the positive cells an option?
I can think of how if some of the cells that are degrading g418 allows for untransformed cells to surive, or they could kick out the plasmid of your gene and keep the g418 cassette.
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