I have to do the migration assay and I have subcultured the cell (cell count = 920000/ml) and viabilty is 96.5%. Could anyone suggest possible protocols for this as I have searched but didn't find anything consistent?
Hi Prem
Mentioned in this article:
IGFBP-5 induces cell adhesion, increases cell survival and inhibits cell migration in MCF-7 human breast cancer cells.
PMID: 22328518
Hi Prem,
It depends on the type of wounding you are conducting. Will this be scratch or electric-induced injury via ECIS which provide a uniform wounding of the cellular monolayer (250um in size). You can also conduct mechanical injury with a sterile toothpick (circular shape) see one of my published article, doi:10.1152/ajplung.00373.2005
Am J Physiol Lung Cell Mol Physiol 290:L849-L855, 2006. Hope that would be helpful.
Diane
Hi Perm,
I suggest you ECIS instead of scratch. Please note that it is very hard to have standard scratches therefore the evaluation is also very hard.
In ECIS there are two choices for wound healing:
(i) to form the wound by electric current - this will remove the cells from the surface of the spot like gold electrode. In this case after wounding you can follow the change (increasing) of impedance as a good mirror of migrating cells onto the cell free spot.
(ii) the other choice to use "fence" mode of ECIS - in this case you can turn on a "fence" like electric field which will keep the electrode free of cells in the first period of cell culturing; after that - whithout any electric shock etc. you can turn off the fence and the cells will start to migrate into the cell-free zone.
There is a third nice choice if you have no choice to use ECIS (if you want I can offer you our collaboration) -
this is an easy tool "culture insert" made by ibidi (Germany). This provides a standard wound. Details you can find on www.ibidi.com, the cat. No. is 80206 and some others depending on the coating.
Good luck!
(If you need more information you can find me on the [email protected])
Platypus (Oris) migration assay could be used. It is a 96-well plate format where wound size is defined by the silicon insert. You plate your cells and when they rich high confluency (become a monolayer) you remove the insert, creating free space circular wound.
Ibidi has some convenient plates for would healing experiments. I have used the 35mm ones. I plated 5 105 cells into the plates and let them overnight to reach confluency in serum medium. The next morning I took out the culture insert and observed for around 20 hours. I guess though, that in the case of MCF-7 the number of cells and the hours needed for feeling the gap could be less. I have heard that the scratch of plates with tip works well too but I have never tried it to be honest.
- Badges
- Science topic