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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!

 

-nazli-

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

-bob1-

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?

-nazli-

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.

-bob1-

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

-nazli-