Hi,
I am trying to create a stable cell line with my protein of interest inserted in vector expressing GFP. However my protein is not directly attached to GFP expressing region.
I would like to know after transfection, should I directly go with FACS cell sorting or do I need to first grow the cells for sometime in G418 containing medium before proceeding with FACS.
As Sima indicated above you may go for selection before sorting if your plasmid has a selectable marker. If your plasmid does not contain a selectable marker the cells will begin to loose the GFP signal.
Preferably, I will go for bulk sorting 48 hrs after transfection if my plasmid does not contain a selectable marker.
Also, if the transfection efficiency is very poor sorting the cells after transfection and plating in smaller well plate (depending on the total amount of GFP positive cells sorted) will be good for cells proliferation. Especially cells that do not grow well in very sparse conditions.
@Sima Lev thanks for your input. Last time I did selected single colony with G418 and proceed with further maintaining the cells, however with time I do see fluctuation in my protein level of expression so I am confused as it had polyclonal population and thus resulting in completing results.
Thats why thought of whether FACS sorting will be of some use for selecton.
I will go for cell sorting 48-72 hours after transfection if you are interested in monoclonal cell lines. You can now continue selection with G418
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