Transfection & Wound Healing Assay - (Aug/06/2008 )
I am trying to set up some migration/invasion assays to assess the effect of overexpression of my gene of interest on the ability of various cancer cells to metastasize. I was thinking of starting with the wound healing assay as it seems easy enough & inexpensive. I was just wondering what do other people do with regards transfecting their cells. I was thinking of transfecting my cells first and then about 24hrs post transfection wounding the cells with a pipette tip. Or would wounding the cells & transfecting them simultaneously work better? (I tried this earlier and it seems that cell death due to lipofectamine seems to affect the wound hence i was considering inflicting the wound post transfection). What do you think?
Also I usually grow all my cells with 10% serum. Naturally this amount of serum would influence the wound healing response so i was going to try to serum deprive the cells for at least 6hrs before inflicting the wound and then leave them in serum free media. Does this sound ok?
All feedback greatly appreciated!
The cells do need some time to transcript the transfected gene and to produce the protein you wish to examine. You can create a wound between 12- 24 hrs. Your transfection can be done in the presence of 2-5% serum in Opti-MEM with lipo2000, then you can change it to a complete medium after 5-6 hrs, then do the serum starvation for 6 hrs and introduce the wound afterwards. No entirely sure about the component of Opti-MEM, therefore you may want to avoid it in the last step.
Excellent! Thanks for that.... much appreciated!