In vitro Cell Migration, Wound Healing Full Protocol

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What is Cell Migration?

Cell migration, movement of cells from one area to another generally in response to a chemical stimulus.

Migration is a central to achieving functions such as wound repair, cell differentiation and embryonic development, immune response, and disease processes such as cancer metastasis and inflammation.

Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology

Principle of Cell Migration

Migration assay is commonly used test to study the migratory response of endothelial cells or tumor cells to tumor promoters or inhibitors.

In migration assay the cells are seeded and when it reaches a confluency of 90% a scratch is made to mimic a wound.

This wounded cells are exposed to different concentration of test compounds to check its effect on the migratory property of cells.

Cell Migration Assay Requirements

1. Cancer cell line

2. Complete growth media

3. 1X PBS (pH-7.2)

4. 6-well plate

5. Micro-tips

6. Pipettes

7. Light Microscope

8. CO2 incubator

9. Biosafety cabinet level II

Procedure of Cell Migration Assay

1. Seed appropriate number of cells (e.g. for MDA-MB-231 seed approx. 2.5×10^5 cells per well) in 6 well plates.

Make duplicates for each treatment and control group. Incubate in 5% CO2 incubator for 24-36 hours until they reach ̴ 90% confluence.

2. When the cells are ̴ 90% confluence, make a scratch with 200 μL tip in the middle of each well and flush the scratch area with 1 ml pipette gently, so as any of adhered cells from the scratch area get detached.

3. Discard the media wash with 1 ml PBS and add freshly prepared treatment media.

4. Take the images at 0, 12, 24 and 48h, as per the requirement of your experiment (Observe the cells before taking images each time, sometimes some percentage of cells at higher doses get died or undergo apoptosis after 12h or 24h.

These cells come out in the scratch area and we get blurred image of the scratch area with undefined boundaries.

In such case, first collect the treatment media in separate tubes, wash with PBS and keep PBS in wells while taking images.

After taking images, replace PBS with treatment media).

5. Arrange the representative images for each treatment group and perform ImageJ analysis to measure the scratch area and represent your results as percent migration area or wound closure area.

Cell Migration Assay Precautions

Do not use non sterile tips to make the scratch.

Make the scratch straight with steady hands otherwise the wound will not be clear and the cells will be disturbed which will result in insignificant data.

Always take the image of area which shows least dead or floating cells to produce significant and publishable data.

Cell Migration Citations:


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