Hi,
I am working with a slow proliferative cell line (doubling time 18-20 hours) and I want to perform cell cycle analysis by FACS after transient transfection with my protein of interest. I did the experiment once and I found mainly the cell population is in G1 phase(90%). so I decided to do cell synchronization first after 24 hours of transient transfection and then perform CC analysis.
But I am not really sure what would be the best method as the protein I am working with is really unstable and has low transfection efficiency. The cell line I am working with never reaches full confluency and they show contact inhibition.
Any idea would be helpful!
Thanks.
Hi Tanmoyita, the cells with doubling time of 18-20 hours are really fine, not that much worse as you thought. However we need to know some details regarding your first try. For example: which transfection you performed? over-expression or siRNA-mediated knockdown? how many hours you did for the transient transfection? Did you measure the target protein? It is very important. If your protein was not changed, which means your transfection did not work at all.
Hi Han, Thank you for your answer! to answer your question:
I overexpressed my protein by Fugene, I fixed the cells for intracellular staining after 24 hour of transfection (might be short). I detected the protein but the positive population was like 10% and the majority of the cells were in G1 as I said.
I worked with HEK before which gave me promising result but then again I am a bit concerned about using HEK for CC analysis.
Hi Tanmoyita, 24 h-transfection is a little bit short, have you tried 48 h? Another possible reason is: if the target protein is unstable, as you mentioned, it might be degraded very soon, it's difficult to evaluate its functions. You need to find a way to stabilize this protein, or try to enhance the transfection efficiency.
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