Cell adhesion assay - (Oct/05/2006 )
I need to perform a cell adhesion assay where I look at breast cell (ie. MCF7, T47D) attachment to ECM components or attachment to each other. I want to overexpress a protein that may decrease adhesion and compare to cells that are not overexpressing the protein. Does anyone know of a good protocol to perform this study? Thank you.
first, it should be clear what the qualitiy of the cell-cell-adhesion is; adenocarcinoma have defects in building epithelia; if there are molecular markers known, f.i. for adhesion junction such as E-cadherin or catenins, or tight junctions such as ZO-proteins, one has to look for their intercellular expression (immunocytochemistry), or if as GFP-trans/infected-fusion proteins with fluorescence microscopy
for cell-ECM adhesion you may perform video microscopy (changes of cell shape and/or motility) or motility test; there is a good motility test described with matrigel on this side in the "protocol" section; you may check phosphorylation status of FAK, paxilin or CAS by Wb
If you're just looking for the total number of cells which have attached after a certain time, you could assay the DNA after washing your substrate to remove unbound cells.
Of course, this won't differentiate between the cells that are bound to each other and the cells that are bound to the material - though if you're not using a high density of cells, this will be less of a problem.