Question

You are performing a wound-healing assay in the laboratory.

a) What protein would you use to coat the cell culture dishes and why?

b) What effect on wound healing would you expect if you were examining cells that had a mutation in the talin binding domain of integrins?

Answer 1

Wound healing assay, also known as scratch assay or scratch wound healing assay, is used to for analyzing cell to cell interactions and cell migration. This assay can also be modified to observe, effect of chemicals on cells, growth, and cellular proliferation, such as in metastasis.

The basic concept of wound healing assay includes:

1) Making a scratch on the monolayer of cells,

2) This results in a ‘wound gap’,

3) the rate of healing of this gap is monitored (image capturing) and quantified, as the migratory and growth cells moves towards the wound,

4) The increase or decrease of motility or growth rate relates to the factors affecting the process.

a) To conduct wound healing assay, it is necessary to create a confluent monolayer of cell culture on culture dishes. It is important that the cells adhere to the surface of the cell culture plate and does not peel off or dethatch.

Thus, the plates should be coated with extracellular matrix (ECM). ECM can act as glue or adhesive, to hold the cells on the plate. Example fibronectin, vitronectin.

b)

) To maintain the intricate structure of tissues, it is important that there is proper adhesion between cells ant their extracellular matrix (ECM).

Integrins help in cellular adhesion to ECM. Integrin comprises of adhesion receptors which can mediate cell adhesion and migration on extracellular matrix. This plays an important role in wound healing. Talin mediates the association of integrin, through intracellular adhesion complex. The head domain of Talin, binds to the Beta-tail subunit of integrin, resulting in activation of integrins.

If Talin binding domain (beta subunit) of integrin has mutation, it cannot bid with Talin and cannot be activated. This will be reflected as decreased rate of wound healing, in the assay.