need help for transfection to synchronized cells - (Mar/31/2014 )

Hi everyone,

I'm having a problem with transfection to synchronized cells.

My aim is to express a GFP-tagged protein  and see its localization during mitosis. Actually I know It localizes to cell membrane during mitosis and I've already succeeded to  see this localization. However, i need to have a high frequency of cells which are transfected and in mitosis in the same time.

I'm using lipofectamine transfection and my transfection frequency is not so bad. Also i tried double thy and single Thy+STC synchronization methods to synchronize cells and they both seem to work good as well (without transfection protocol)

However when i try to synchronize and transfect the cells, i can not have the sufficient amount of cells that are in mitosis and expressing my protein.

Any suggestions would help! Thank you!

Have you tried the other way around - transfect and then synchronize?  Could you do some FACS to get sufficient cell numbers?

bob1 on Mon Mar 31 20:05:47 2014 said:

Have you tried the other way around - transfect and then synchronize?  Could you do some FACS to get sufficient cell numbers?

Thank you for reply!

I've thought that however according to protocol, I get optimal transfection in 48th hour. In double thy, synchronization takes about 52 hour so I started to synchronize cells than, in between, I made the transfection.

On the other hand in single Thy+STC, i had to transfect first and than start to synchronization procedure (according to needed time intervals for each procedure)

I haven't used FACS before, accually I'm not really familiar with it. Is it possible to use sorted cells for further analysis after FACS?

Most transfections with plasmids will still be expressing fairly well 72 hours or more after transfection (assuming the product isn't toxic).

FACS is fluorescence activated cell sorting - it has a device that can sort and collect the cells based on characteristics you define - which in your case would be presence of a fluorescent marker.  This is not just basic flow cytometry and requires a specialist machine.

Unfortunately we don't have the FACS, thank you for recommadations.