Cellular wound healing assay

Summary

The cellular wound healing assay is widely used and improved by researchers to study the role of various experimental conditions such as gene knockout and chemotherapy in cell migration and proliferation. The advantages of this experiment are (1) simple to perform (2) inexpensive (3) experimental conditions can be easily improved for different purposes.

Operation method

Cellular wound healing assay

Principle

Scratches are made between monolayers of cultured cells to produce healing areas, and then the migration of cells around that wound toward the scratch, i.e., wound healing, is monitored. Factors that alter cell migration and/or growth can increase or decrease the rate of wound healing. The method can be quantified and is suitable for automated systems for high throughput screening.

Materials and Instruments

Human MDA-MB-231 Cells
DMEM medium Fetal bovine serum PBS Glutaraldehyde Ethanol Crystalline violet
24-Well Plate Tip Cell Culture Instrument

Move

1. Cells are grown in DMEM medium containing 10% FBS.

2. Cells are inoculated into 24-well cell culture plates at a certain density, and after 24 hours of growth, the monolayer should reach 70-80% cell fusion.

3. Do not change the culture medium. Use a new 1ml tip to gently scratch between the monolayer of cultured cells, the scratch goes across the wells, and the tip is as perpendicular as possible to the bottom of the plate wells, not tilted. The distance of the gap is equal to the outer diameter of the end of the tip, which can be adjusted with different types of tips. Scratches in the same direction into a straight line.

4. Make another scratch in the direction perpendicular to the first one, and make a cross shape for each hole.

5. After scratching, gently wash the plate wells with culture medium 2 times to remove the shed cells.

6. Add fresh medium to each well.

7. (The medium contains certain ingredients such as chemicals that inhibit/promote cell migration and/or proliferation)

8. Cells are grown for 48 hours (or as long as needed).

9. 1xPBS washes the cells twice, then fixes them with 3.7% paraformaldehyde for 30 minutes.

10. 1% crystal violet (dissolved in 2% ethanol) staining for 30 min.

Caveat

In order to reduce the variability of the experimental results, it is recommended to select more than one field of view per well for observation and do more than one replication per group.

Common Problems

The distance of gap can be measured by Photoshop or ImagJ software, and in order to reduce the variability of the experimental results, it is recommended to choose more than one field of view to observe per well, and do more than one repetition per group.

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