My work is on in vitro transcription of some membrane molecules and transfection of these molecules to the Dendritic cells. Dendritic cells have these molecules and I can not evaluate the correction of my steps in these cells. My method for evaluation of these membrane proteins is flocytometery. I want to evaluate transfection of in vitro synthesized mRNA to a cell that doesn’t have these molecules (CD40, ICOSL, PDL1, PDL2 …) or at least had low levels of these molecules.
So
What kind of cell do you suggest to me?
I have cell lines (4T1, HEK,..) , but my concern is high level of their proliferation and may cause decrease in expression due to division ?? (transfected will not be replicated and division time is less 24-48-72 hr for evaluation of membrane expression of molecules)
Can I stop cell division by Mytomicine C ? This drug will not affect protein membrane expression?
Any other kind of evaluation or advice?
Best regards
You can check the transfection looking for the protein on a wb, immunofluorescence or flow cytometry (as you already use). To your concern about the division time, you could lower the concentration of serum on the medium to 2-3%. I work with 293T, which are great to protein expression, but indeed they divide very fast. Can't you analyze protein expression in smaller time periods? Like 24 hs or 12hs? Alternativelly, you can transfect the cells with plasmids encoding these proteins and check the level of transcription by qPCR.
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