Wound Healing Assay Protocol (2023)

Wound Healing Assay Protocol (1)
Wound Healing Assay Protocol (2)

This protocol helps to set up a Wound Healing Assay using HoloMonitor® M4 and the HoloMonitor® App Suite software. The HoloMonitor® Wound Healing Assay provides an automated label-free wound healing assay, measuring gap closure. Additionally, selected cells can be individual cell tracked for detailed individual cell movement and morphology analysis.

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  • HoloMonitor® M4, placed in incubator
  • HoloMonitor® M4App Suite
  • Culture vessel of choice with cells
  • HoloLid™ for selected vessel
  • Vessel holder for selected vessel


  • Gap width (μm)
  • Cell covered area (μm2 and %)
  • Cell free area (μm2 and %)


  • Guided assays
    • Cell Quality Control
    • Kinetic Cell Proliferation
    • Kinetic Cell Motility
    • Kinetic Dose Response
  • In-depth analysis
    • Single Cell Tracking
    • Cell Morphology



  • HoloMonitor® M4, placed inside the incubator
  • HoloMonitor® App Suite software
  • Cell culture vessel. Please check our list of recommended vessels.
  • HoloLid™ for the selected vessel
  • Vessel holder for selected vessel
  • Setup and Operation Manual for HoloMonitor® M4
    ► We recommend using ibidi® µ-Dish 35mm with Culture-Insert 2Well high (cat. # 81176) or ibidi® µ-Plate 24-well with Culture-Insert 2Well (cat. # 80241)


Wound Healing Assay Protocol (3)

Seed the cells to at least 90% confluence in the inserts according to the ibidi protocol.

Place the vessel in the incubator and let cells attach for 2-24hours.

Start the software and wait for complete instrument initialization.

Run an auto-calibration. With successful calibration (as shown in the picture), the instrument is ready to use.

Wound Healing Assay Protocol (4)

Sterilize the HoloLids™ according to the HoloLid™ sterilization and use protocol.

Add the treatment to your cells. The final working volumes per well, essential for using HoloLids™, are shown in the table above.

Slide the cell culture vessel onto the Vessel holder, its grips facing towards you. Ensure that the vessel is parallel to the holder. There is a spring that holds the vessel in place.

►When using multi-well plates, place them with the cut-off corner to the left.

Replace the standard lids with the HoloLid™.

Put the vessel holder with the sample on the HoloMonitor® M4stage.

Select the Wound Healing Assay and proceed by clicking the Setup Capture button.

(Video) Wound healing


Basic setup: Describe the experiment and assign treatments to the wells

Wound Healing Assay Protocol (5)

Enter the experiment name, optional experiment
and cell types.

Select the correct vessel map from the drop-down list.

Map treatments and conditions on the vessel map. Select wells by marking them with the left mouse button while moving the cursor over the well/s.

Add the treatment name/s in the text box below the vessel map and click Add/ press Enter. Marked well/s are light blue, selected wells will appear dark blue.

Proceed to Capture setup.

Capture setup: Select the experiment time settings and choose capture positions

Wound Healing Assay Protocol (6)

Adjust the default settings for duration and interval.

Add capture positions: The position list is open by default. Click positions on the vessel map and add them to the position list with the Add current location button. In case the image quality is poor, a warning sign appears. Adjust focus or position location if necessary.

(Video) Scratch Assay Tutorial

►Note that the gap might not be exactly where the vessel map indicates.

Ensure that the storage requirement for the experiment does not exceed the computer capacity.

► When running an experiment, data needs to be stored on the computer connected to the instrument. Storing data on an external drive (e.g. connected via USB or internet server) may cause data loss due to erratic USB connections or poor internet connection.

Wound Healing Assay Protocol (7)

Run a full or quick validation of the selected positions to ensure good image quality.

When satisfied with the experiment setup, click Proceed to Capture.

►If there are no positions with both wound edges visible at the same time, add two positions that are parallel to each other and have one edge with cells. Use XY position Arrow buttons to move the stage in steps and when satisfied, press Add Current Location button.

Capture: Review the experiment in real-time during the time-lapse

Wound Healing Assay Protocol (8)

Click Start Capture.

To stop the experiment ahead of time, click the stop button.

► Note that it is NOT possible to restart the experiment once it has been stopped.

Go to the Experiments tab and open your ongoing experiment to preview the results during the run.

► Wait for the experiment to finish before starting In-depth Analysis.

When the Experiment capture finishes, click the Show Result button to get directly to the Results page.


Experiments tab

Wound Healing Assay Protocol (9)

Click Experiments to see a list of the experiments.

(Video) Wound Healing Assay | Scratch Assay |

Click on the experiment title to open an experiment summary.

Click Open to open the results page.

Experiment overview tab

Wound Healing Assay Protocol (10)

See the experiment summary, view all images, and go to the experiment setup by choosing the respective tab.

Generate in-depth analysis data from the captured images by clicking on the Wound Healing icon. A new window for the in-depth analysis will open.

Create New Guided Assay Results from this experiment by clicking the respective button.

In-depth analysis —Wound healing tab

Wound Healing Assay Protocol (11)

Begin by adding frames to the analysis either by drag and drop or using the Add selected or Add all button.

►Check the image quality before using it for in-depth analysis. See the Image quality guide for more information.

Adjust the image threshold and object size and click Apply to all frames. You can alter the viewing options.

Wound Healing Assay Protocol (12)

Based on the identification in the previous step, result values are generated and displayed in the table: gap width (mm), cell-covered area (% and μm2), and cell-free area (% and μm2).

►In case of outliers, select the outlier frame in the list and adjust the threshold for that specific frame.

Check the gap width and cell coverage graphs to ensure that the values decrease evenly with time.

Export to Excel for further analysis. The exported data include gap width (μm), cell-covered area (% and μm2), and cell-free area (% and μm2), graphs for gap width, cell coverage, and the settings overview.

►From the results, it is easy to get the speed of the cell front (cell front velocity).

►If there was only one edge with cells visible in a field of view. To evaluate gap closure kinetics, calculate cell front velocity for each edge with cells and assess cell covered area (%) change in the field of view.

(Video) Wound Healing Assays Using the Culture-Insert 2 Well

Wound Healing Assay Protocol (13)

►Calculate the speed of the cell front by subtracting the last value for gap width in the linear phase from the first value for gap width within the linear phase and divide with the number of hours between the values.

►In this example: (321-25) μm/36h = 8 μm/h
This can also be performed in the Excel datasheet.

►Note that it is important to use only data from the linear phase of the slope.

One experiment — multiple results

  • This section helps to reanalyze data between different assays using HoloMonitor® App Suite software.

Generating In-depth Assay results

In the Experiment overview page select the In-depth application icon for wanted result.

Follow the respective assay protocol.

Generating Guided Assay results

Wound Healing Assay Protocol (15)

In the Experiment overview page under Guided Assay Results select Create New Result.

Choose the type of analysis in the pop-up window and name the new result.

Tick copy image analysis from and select the experiment to copy from. This will copy the image analysis settings from the selected result including all changes.

► For further data analysis steps, please see the respective assay protocol.

Press create.

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(Video) Scratch Migration Assay and Skinfold Chamber: Wound Healing Analysis | Protocol Preview


How is wound healing assay calculated? ›

Where Wi is the average of the initial wound width, Wf is the average of the final wound width both in μm and t is the time span of the assay in hours. Additionally, At = 0 is the initial wound area, At = Δt is the wound area after n hours of the initial scratch, both in μm2.

What are the limitations of scratch assay? ›

When the scratch is done manually, it's susceptible to 'ragged' edge boundaries, which make analyzing data more difficult. Also, the damage could physically damage the cells adjacent to the wound and create inaccurate wound size areas. This limitation is slowly becoming less of an issue with automated technologies.

How to do a scratch wound assay? ›

Scratch assay on non-transfected cells

Scrape the cell monolayer in a straight line to create a “scratch” with a p200 pipet tip. Remove the debris and smooth the edge of the scratch by washing the cells once with 1 ml of the growth medium and then replace with 5 ml of medium specific for the in vitro scratch assay.

When conducting a wound healing assay how do you ensure that wound closure is due to migration and not proliferation? ›

However, the cells density is higher after 16h indicating that the cells are overcrowding. For migration assay, you should use mitomycin C to suppress cell proliferation to make sure that the wound healing is because of migration and not the cell proliferation.

How do you quantify wound healing? ›

The first method measures the change in the wound width (nanometres) over time. This width is the average distance between the two margins of the scratch. The second method calculates the change in wound area over time as a percentage of wound closure. These two methods can be time-consuming when performed manually.

How do you complete a wound assessment? ›

  1. Identify the wound location.
  2. Determine the cause of the wound:
  3. Evaluate for foreign bodies or neoplastic processes. ...
  4. Determine the stage of the wound:
  5. Stage I: Superficial, involving only the epidermal layer. ...
  6. Evaluate and measure the depth, length, and width of the wound[51]

How do you quantify cell migration? ›

Migration can be assessed by determining the number of cells that move across a microporous membrane (transwell migration assay) or by measuring the surface area that cells occupy over time after creating a 'cell-free' area (scratch assay) [8–10].

What is scratch assay use for? ›

The scratch assay is often used to test cell migration because it is cheap and easy to perform on adherent cell lines, such as fibroblast, endothelial, and epithelial cell lines6,7.

What is an invasion assay? ›

The Cell Invasion Assays offer a flexible, standardized, high-throughput format for quantitating the degree to which invasive cells penetrate a barrier consisting of basement membrane components in response to chemoattractants and/or inhibiting compounds.

What are 4 components of a wound assessment? ›

Tissue Loss. Clinical appearance of the wound bed and stage of healing. Measurement and dimensions. Wound edge.

What are the 6 key principles of wound assessment? ›

The basic principles for the management of a wound or laceration are:
  • Haemostasis.
  • Cleaning the wound.
  • Analgesia.
  • Skin closure.
  • Dressing and follow-up advice.

What are the 5 important reminder that you need to follow in wound dressing? ›

The overall objective of a wound dressing include the following:
  • Decrease the pain.
  • Apply compression for hemostasis.
  • Protect the wound from the environment.
  • Protect the wound from soiling with body fluids or waste.
  • Immobilize the injured body part.
  • Promote wound healing.

What are the five critical elements that need to be addressed before effectively preparing a wound bed to heal? ›

Abstract. This paper explores the concept of wound bed preparation and relates this to five key areas of care: bacterial balance, necrosis, exudate, cellular dysfunction and biochemical balance.

What are the 4 methods of wound closure? ›

Wounds can be closed primarily in the emergency department (ED) by the placement of sutures, surgical staples, skin closure tapes, and adhesives.

How do you calculate epithelialization? ›

In each wound, the percentage of wound epithelialization was calculated by the following formula: 1 − (pixels open wound area/pixels total wound area) × 100%. Fig.

What lab value is important for wound healing? ›

Important parameters to evaluate include protein levels, complete blood count, erythrocyte sedimentation rate, liver function tests, glucose and iron levels, total lymphocyte count, blood urea nitrogen and creatinine levels, lipoprotein levels, vitamin and mineral levels, and urinalysis.

What is the most common method for wound measurement? ›

The most common method is linear measurement, also known as the "clock" method. Imagine the body as the face of an imaginary clock, the head is 12:00 and the feet are 6:00. Length is measured by placing the ruler at the point of greatest length or from 12:00 to 6:00.

What is wound healing index? ›

This index assesses wound healing using scores from 1 to 5: a wound with very poor healing receives a score of 1, whereas excellent healing receives a score of 5. A further index, the Early Healing Index (EHI), was developed by Wachtel et al. [10], classifying healing in 5 degrees.


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