Dear Dr. Roghayeh Mokhtari Asl,

In the wound healing assay, the scratching process may involve mechanical injures to the cells at the wound edges. It exerts high shear from the wound edges influencing cell-cell as well as cell matrix interactions, where the fibroblasts need to re-establish through cell-surface interactions prior to accelerating migration into the defective cell-free area. Some damaged cells and cell debris can also get attached to the wound margins, perturbing the motility of other cells.  Additionally, the migrating surface can be damaged in the scraping process, leading to preferential paths in cell movements. The leading cells disrupt their site of adhesion, protrude, and contract the cytoskeleton to move.

So, you will observe some changes in cell morphology, but they maintain the original shape, with round pseudopodia, and the group of cells move forward uniformly making the wound margin look dim. Please note that depending on the cell type, they can migrate as individual cells (for instance, fibroblasts) or collectively as sheets of cells (for example, epithelial and endothelial cells). Usually, 8-24 hours is needed for cells to close the scratch depending upon the cell type used.

From the image it seems to me that the wound has not healed even at 24 hrs after the scratch.

I have a few questions.

1. Do you have a control image (cells without treatment)?

2. How do you introduce the scratch?

Usually, a scratch is created with a sharp object such as a pipette tip or a needle. The best way would be to use a 20ul tip. Keep it straight without twisting, applying constant pressure.

3. Is your treatment aimed to hasten or slow down the healing process?

Regards,

Malcolm Nobre

Hello

Thank you very much for your response

I took pictures of the cells from 0 to 96 hours. In this treatment, the photo of which I have attached, the cells remained the same from 24 hours onwards and the wound area was no longer visible.

I am attaching a photo of the control group.

I performed the scratch with Tip 20.

My ultimate goal is to study wound healing on an animal model and before that I want to measure cell migration with a scratch test. i also attach T48 and T96 of this treatment.

The cells morphology has not changed much, rather their orientation is now towards the wound gap, and they are attempting to fill it/ grow in that region.

You can stain for Focal adhesions before and after scratch assay and characterize their formation at the edges, you will find a change in the number and positions of the formation of these focal adhesions. It depends on what the aim of your experiment is. Some data on the actin cytoskeleton and focal adhesions will support your wound healing study.

Shayan Singh Parihar

I am very grateful for your response.

My goal with this experiment is to investigate the migration of cells to the wound area.

If possible, could you explain more about the focal adhesion staining or if you have an article, please send me a link to it.

Thanks.

Article Analysis of Focal Adhesions and Cytoskeleton by Custom Microarray

https://star-protocols.cell.com/protocols/2937

Article Step-by-step quantitative analysis of focal adhesions

https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/155/976/fak100.pdf

However, this is more on the cellular level, cell migration can be simply characterized from the gap of scratch by ImageJ