Scratch Assay Migration vs Proliferation: Is Your Wound Closure From Cell Proliferation Rather Than Migration?
- CLYTE research team
- Jul 11
- 5 min read
One of the most persistent challenges in wound healing research is deciphering the true nature of wound closure in a scratch assay. Are your cells genuinely migrating into the created gap, or is cell proliferation clouding your results? This critical question can determine the validity of your findings. Fortunately, there's a powerful tool to provide a clear answer: Mitomycin C.
When studying the effects of growth factors, testing novel drugs, or evaluating biomaterials, distinguishing between cell migration and proliferation is paramount. If cells are dividing, you can't be certain that the observed wound closure is due to active cell movement. This is where Mitomycin C becomes an indispensable reagent in your experimental toolkit.
You might also be interested in this Scratch assay SOP!
You might also be interested in this Crystal violet staining for scratch assay!
What is Mitomycin C and How Does It Stop Cell Proliferation?
Mitomycin C (MMC) is a potent antitumor antibiotic that functions by crosslinking DNA. This action effectively halts DNA replication, thereby inhibiting cell division. When used at low concentrations and for short durations in a scratch assay, MMC can stop proliferation without inducing significant cell death.
By incorporating this DNA crosslinking agent into your protocol, you can effectively isolate and measure pure cell migration. This ensures that your results accurately reflect the motile properties of your cells, unmasked by the confounding factor of proliferation.
A Step-by-Step Protocol for Integrating Mitomycin C into Your Scratch Assay
Timing and concentration are critical for obtaining reliable, proliferation-independent data. Follow this detailed guide for seamless integration of Mitomycin C into your scratch assay workflow.
1. Preparation of Mitomycin C Stock Solution
Stock Concentration: Prepare a 0.5 mg/mL stock solution in sterile water.
Dissolving: Using sterile techniques, dissolve the Mitomycin C powder. It is highly sensitive to light, so handle it in dim lighting or use foil-wrapped containers.
Sterilization and Storage: Filter the solution through a 0.22 µm filter. Store the aqueous solution at 2–8°C, protected from light, and use it within one to two weeks.
Labeling: Always label your stock solution with the concentration, preparation date, and expiration date. Discard the solution if you observe any precipitation or discoloration.
⚠️ Safety First: Mitomycin C is cytotoxic and a suspected mutagen. Always wear appropriate personal protective equipment (PPE), including gloves, a lab coat, and safety goggles.
2. Cell Seeding and Monolayer Formation
Seed your cells in a multi-well plate (a 48-well plate is a common choice) at a density that allows them to reach 100% confluency.
Incubate the plate at 37°C with 5% CO₂ overnight, or until a complete monolayer has formed.
3. Mitomycin C Treatment (Pre-Scratch)
Working Solution: Prepare a working solution of 5 µg/mL Mitomycin C. Depending on your cell line and experimental design, this can be in a serum-free medium or a medium containing 1% Fetal Bovine Serum (FBS).
Incubation: Aspirate the existing medium from the wells and add the Mitomycin C working solution. Ensure the plate remains protected from light. Incubate for 2 hours at 37°C.
Washing: After the incubation period, carefully remove the Mitomycin C solution and wash the cells thoroughly with Phosphate-Buffered Saline (PBS) to eliminate any residual MMC.
Final Medium: Replace the PBS with a fresh, serum-free medium. Your cells are now viable but will not proliferate during the assay.
4. Creating the Scratch
Using a sterile 200 µL pipette tip or CellCut, create a straight, uniform scratch through the center of the cell monolayer.
Gently wash the wells with PBS or your medium to remove any detached cells and debris.
Add your experimental treatments (in serum-free medium) or a control medium to the wells to initiate the migration phase.
5. Imaging and Data Quantification
Immediately capture an image of the wound area (this is your 0-hour time point).
Continue to image the scratch at regular intervals (e.g., 6, 12, 24, and 48 hours) to monitor wound closure.
Quantify the rate of wound closure using image analysis software such as ImageJ. See this article
Pro-Tips for a Successful Assay
Optimization is Key: The ideal concentration and incubation time for Mitomycin C can vary between cell lines. It's crucial to optimize these parameters for your specific cells.
Include Controls: Always run a control group without Mitomycin C. This will allow you to directly compare wound closure with and without proliferation, highlighting the impact of cell division.
Use Serum-Free Media: After creating the scratch, use a serum-free medium to avoid introducing additional factors that could stimulate proliferation.
Thorough Washing: Ensure all residual Mitomycin C is removed after treatment, as any remaining MMC could negatively impact your results.
Essential Safety Precautions
Handling: Mitomycin C is cytotoxic and mutagenic. All handling should be performed in a biosafety cabinet or a fume hood.
Waste Disposal: Dispose of all waste contaminated with Mitomycin C according to your institution's guidelines for hazardous chemical waste.
Personal Protective Equipment: Always wear a full complement of PPE, including gloves, a lab coat, and eye protection.
Light Sensitivity: Keep all stock and working solutions of Mitomycin C protected from light to maintain their efficacy.
Final Thoughts
If you want to be sure your scratch assay reflects true cell migration, not a blend of movement and mitosis, Mitomycin C is your best friend. A simple pre-treatment step turns your assay from ambiguous to precise—making your conclusions more reliable and publication-ready.
It’s a small tweak with a big impact on data quality.
Frequently Asked Questions (FAQ)
1. Does Mitomycin C kill the cells?
Not at the right dose. It inhibits proliferation but leaves most cells metabolically active and viable—especially at 10 µg/mL for 2 hours.
2. Can I use Mitomycin C after scratching?
No. MMC is most effective before the scratch. Post-scratch treatment may interfere with migration or wound healing dynamics.
3. What medium should I use during treatment with Mitomycin C?
Serum-free or low-serum medium is preferred to avoid cell division stimulation during MMC incubation.
4. Can I reuse Mitomycin C stock solutions?
If aliquoted and frozen properly, yes—but avoid multiple freeze-thaw cycles. Discard any solution showing color change or precipitation.
5. How do I confirm Mitomycin C worked?
Compare your MMC-treated assay to a non-treated control. If the scratch closes slower or not at all, MMC likely suppressed proliferation.
6. Can I combine Mitomycin C with Live/Dead or Crystal Violet staining?
Yes! You can use MMC to block proliferation, then follow with staining to visualize migration and viability more clearly.
7. Is Mitomycin C always strictly necessary in a scratch (wound healing) assay?
N0! The decision to use MMC depends on the experimental question:
- If you are interested in pure migration (excluding proliferation), MMC or another proliferation inhibitor should be used.
- If you are studying wound closure as a combined effect of migration and proliferation (e.g., in tissue repair models), you may choose not to use it.
- Also, for short-term assays (evaluating migration after 6h) or with slow-growing cells, proliferation may have minimal impact, and MMC is not required.
