Wound healing assay
Protocol for performing a scratch-based wound healing assay, a type of migration assay.
The wound healing assay, often referred to as a scratch assay, is a widely used method for evaluating cell migration and tissue repair. This wound healing assay protocol offers a guide for conducting a standardized wound healing assay, to study processes such as cell migration, and wound closure. Wound healing assays allow you to generate quantitative data, allowing you to understand cellular dynamics in various contexts, and can be used to assess the effects of novel drugs for various purposes, for example, in reducing cancer migration and metastasis.
Stage 1 - Method
Materials required
- A confluent flask or plate of cells
- 12 well culture plates
- Appropriate growth medium (containing any serum, growth factors or small molecules required for your specific cell type)
- Enzymatic or chemical cell detachment agent (for adherent or semi-adherent cells)
- Standard cell culture consumables (serological pipettes, pipette tips, centrifuge tubes, 70% ethanol, sterile PBS, etc.)
- Any treatments you are assessing
- Standard cell culture equipment (pipette boy, pipettes, containment facilities, centrifuge, light microscope, cell counter/hemocytometer etc.)
- Light microscope with an attached digital camera
- Thin tipped permanent marker
Steps
Wash cells and culture without growth serum
- Using a confluent plate or flask of cells, aspirate the media and dispose.
- Add an appropriate volume of PBS, ensuring the cells are covered.
- Gently swirl the plate, then aspirate and discard PBS.
- Add an appropriate amount of serum-free media (10-12 mL) and culture the cells for 24 h.
Wash and collect cells
- Aspirate media and dispose.
- Add an appropriate volume of PBS, ensuring the cells are covered.
- Gently swirl the plate, then aspirate and discard PBS.
- Add appropriate detachment reagent and incubate at 37 °C until cells are detached.
- Add fresh culture media and transfer cell suspension to a centrifuge tube.
- Centrifuge at 200-250 x g for 5 min.
- Remove media and resuspend the cell pellet in an appropriate volume of PBS.
- Centrifuge at 200-250 x g for 5 min.
- Aspirate and discard PBS.
- Resuspend cell pellet in fresh growth medium.
Any coatings required for culture vessels should be applied prior to cell isolation. Check the repository’s protocols for optimal growth conditions. The protocol is written for the use of the most common detachment reagent (enzymatic); alternative detachment reagents and methods are available.
Seed cells into a 12-well plate in fresh supplemented culture medium
- Transfer the required volume of cells to each well of the 12-well plate such that the cells would be ~80% confluent after 24 h.
- Add supplemented culture medium to the required volume (1-2 mL) and culture the cells for 24 h. Label flasks as required.
- At this point, add any treatments you are assessing if they require a pre-treatment period.
Wound the monolayer of cells
- Using a 200 µl pipette tip, scratch the monolayer of confluent cells in the center and along the entirety of the well's diameter.
Do not be too firm with the tip, as this can result in the plastic residue being transferred from the tip to the plate.
Ensure that the tip makes contact with the bottom of the plate along the entire length of the scratch.
Wash cells
- Aspirate the media from each well and dispose.
- Gently add an appropriate volume of PBS, ensuring the cells are covered.
- Gently swirl the plate, then aspirate and discard PBS. Ensure any detached cells and debris are removed.
- Add an appropriate amount of culture media (1-2 mL).
- At this point, add any treatments you are assessing – assuming they do not require a pre-treatment period.
Image the wound at several points
- Using a light microscope with an attached digital camera, focus on the wound area.
- Ensuring a suitable area of the monolayer of cells is visible on either side of the wound with the wound in the center, obtain an image of the cells.
- Using a thin-tipped permanent marker, carefully mark on the bottom or top of the culture plate where the image was taken.
- Repeat this for each well.
- Return cells to a culture incubator and culture for 24 h.
Re-image the wound at the same points
- Using a light microscope with an attached digital camera, focus on the wound areas marked previously.
- Ensuring a suitable area of the monolayer of cells is visible on either side of the wound with the wound in the center, obtain an image of the cells.
- Repeat this for each well.
- If you are observing wound closure for longer than 24 h, replace media/treatments if required and return cells to a culture incubator and culture for your designated time period.
- Repeat the imaging steps as required.
- Once your assessment is complete, discard of the cells as required by local guidelines and regulations.
Measure wound closure
- Ensure the digital images obtained are named appropriately and filed accordingly.
- Using a tool like ImageJ, measure the width of the wound in the images taken after scratching (a).
- Using the image from the same well after allowing for wound closure, measure the width of the wound following closure (b).
- The percentage of wound closure can be calculated as follows:
- % wound closure = (a-b)/a x 100