Investigating Cell Migration Using Live-Cell Analysis
Cell migration is a fundamental component of many biological and pathological processes such as embryonic development, tissue reorganization, angiogenesis, immune cell trafficking, chronic inflammation, wound healing and tumor metastasis. Extending on the migratory capacity of tumor cells is their ability to degrade the extracellular matrix (ECM) and invade surrounding tissue - a hallmark of cancer and a key factor in metastasis. Understanding the mechanisms involved in tumor cell invasion may lead to limiting tumor progression and a reduction in mortality for many cancer patients. Researchers employ a variety of techniques to investigate and develop novel treatments but are often limited by current techniques which:
- Result in a single, user-defined time point measurement or require manual intervention to generate multiple time points
- Lack precision and reproducibility in creating the wound and capturing data
Scratch assays can be an elegant and convenient alternative to conventional techniques. The Incucyte® Cell Migration and Invasion Assay can quantify migration and invasion potential in the presence of experimental treatments in physiologically relevant conditions in real time. Using the Incucyte® Woundmaker Tool, Incucyte® Imagelock 96-well Plates and the Incucyte® Scratch Wound Analysis Software Module, automated and precise analysis of cell movement can be validated with visual inspection of morphological changes throughout the assay.
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Incucyte® Scratch Wound Cell Migration & Invasion Assay Overview
An integrated solution enables real-time visualization and assessment of cell morphology in scratch wound assays (both label-free and fluorescently labeled) using the Incucyte® Imagelock 96-well Plate. This specially designed microtiter plate contains fiducial marks that allow precise image registration of the same field of view, even when the plates are manually removed and replaced within the Incucyte® Live-Cell Analysis System. These plates are compatible with the Incucyte® 96-well Woundmaker Tool, a 96-pin mechanical device that creates precise, homogenous scratch wounds in all wells at the press of a button. Images are automatically acquired from the same field of view, and the Incucyte® Scratch Wound Analysis Software Module provides real-time quantification of cell migration or invasion through a 3D biomatrix gel. The Incucyte® Imagelock Plate - in conjunction with the Incucyte® Live-Cell Analysis System and Incucyte® Scratch Wound Analysis Software Module - facilitate precise, clear visualization of the initial would and the incremental wound closure at each time point.
Figure 1. Incucyte® Woundmaker Tool
Incucyte® Imagelock 96-Well Plate
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Incucyte® Scratch Wound Analysis Software Module
The integrated analysis algorithm automatically masks each image to identify the position of the wounded (cell-free) and unwounded (cell-occupied) zones, to deliver robust measurements of wound width, wound confluence and relative wound density (RWD) for the entire time-course of the experiment.
Cat. No. 9600-0012
Live Cell Imaging and Analysis by Sartorius
The Incucyte® Live-Cell Imaging and Analysis System allows you to visualize and quantify label-free or fluorescently labeled chemotactic cell migration, invasion and transendothelial migration in real time—directly inside your incubator. Both time-lapse imaging and quantitative data are automatically generated during incubation, providing powerful insights into the time-course of chemotaxis in a physiologically relevant environment. This instrument, in conjunction with the Incucyte® Clearview 96-well Plates and Incucyte® Scratch Wound Analysis Software Module, offers a powerful alternative to end-point measurements and a better understanding of chemotaxis.
Comparing Existing and Incucyte® Scratch Wound Cell Migration and Invasion Approaches
Existing Scratch Wound Assay | Incucyte® Scratch Wound Migration and Invasion Assay | |
|---|---|---|
Migration in the absence of chemotactic gradients | X | X |
Low cell usage (<5,000 per well) | ||
Suitable for non-adherent cells | ||
Migration across a surface | X | X |
Invasion through a 3D gel matrix | X | X |
Integrated quantitation | X | |
Integrated cell observation | X | |
Precision, reproducibility | X | |
96-well throughput or higher | X | |
Kinetic read-out | X | X |
Label-free | X | X |
Workflow: Set up and walk away | X |
Key Advantages of Incucyte® Scratch Wound Cell Migration & Invasion Assay
Label-free quantification of cell migration and invasion - Visualize morphological changes and automatically quantify migration and invasion in real time with the scratch wound assay
See example data below
Investigate treatment effects on migration and invasion – Reproducible 96-well kinetic assay enables pairwise quantification of pharmacological effects
See example data below
Derive deeper biological insights with dual fluorescence readouts – Gain additional understanding of associated biological processes
See example data below
Validated with a wide range of adherent primary, immortalized and tumor cell types
See example data below
Example Data for Scratch Wound Migration and Invasion Assays
Label-free quantification of cell migration and invasion
Visualize morphological changes and automatically quantify movement across a substrate (migration) and through 3D biomatrix gels (invasion) in real time with the scratch wound assay. Robust method allows for kinetic assessment of assay conditions and cell migration profiles.
Figure 2. Quantifying cell migration and invasion with the scratch wound method. HT-1080 cells seeded at 30K cells/well, left for 24h to become confluent and then wounded. HT-1080 cells migrated into the wound over a 12h period, with the segmentation masks showing the initial wound (blue) and the wound closure over time (yellow).
Figure 3. Optimization of assay conditions to control for cell proliferation. MDA-MB-231 and BxPC3 cells were pre-treated with the anti-proliferative compound mitomycin C (MMC) prior to wounding. Images and quantification at 24h show MMC had no effect on MDA-MB-231 cells (top row) and attenuated wound closure in BxPC3 cells compared to vehicle (bottom row). Data indicates proliferation contributes to wound closure in BxPC3 but not MDA-MB-231 cells.
Investigate treatment effects on migration and invasion
Reproducible 96-well kinetic assay enables pairwise quantification of pharmacological effects on cell migration and invasion. Utility and specificity can be investigated alongside images and time-lapse moves to monitor cell morphology changes over time. Scratch wound assays are validated for more than 20 adherent primary and immortalized cell types.
Figure 4. Pharmacological comparison of blebbistatin effects on migratory and invasive phenotype of HT-1080 cells. HT-1080 cells were plated on collagen-1 plated plates and for invasion studies cells were overlaid with collagen-1. Cells were treated with concentration range of blebbistatin. Microplate graph shows migration and invasion over 24h. Time courses and concentration-response curves show concentration-dependent inhibition, with blebbistatin exerting a greater effect on invasion compared to migration.
Figure 5. Comparing migration and pharmacological profiles of glial cells in real time. Migration profiles of primary (Cortex, Hippocampus, Cerebellum) or iPSC-derived (iCell, Fujifilm) astrocytes were assessed. Images show migration of iCell astrocytes over time. Time-course profiles compare rate of wound closure for different brain regions. For iCell astrocytes treated with ionomycin a concentration-dependent inhibition of migration was observed.
Derive deeper biological insights with dual fluorescence readouts
Gain additional understanding of associated biological processes. The ability to image scratch wound assays in two-color fluorescence channels - in addition to phase contrast -allows users to further interrogate cellular processes such as cell cycle progression, and how this pertains to cell movement.
Figure 6. Concurrent measurements of cell cycle progression and cell migration. HT-1080 cells stably expressing the Incucyte® Cell Cycle Green/Red Lentivirus treated with cytochalasin D. Schematic shows colors associated with cell cycle phases (left), fluorescence enables quantification and visualization of cells within the G1 (red) and S|G2|M (green) phase. Green and red fluorescence images reveal inhibition of cell migration (initial scratch wound indicated in blue) as well as a high population of red cells. Time-courses show cytochalasin D has an inhibitory effect on cell migration and the cell cycle through arresting in G1.
Reproducible quantitative measurement without labeling cells
The Incucyte® Scratch Wound Assay has been validated with a wide range of adherent primary, immortalized and tumor cell types, and uses the Incucyte® Woundmaker Tool to create homogeneous scratch wounds per plate in seconds at the press of a button. The unique tip design provides clean, consistent wounds without damaging the cells and creates a cell-free zone from a confluent monolayer of cells making the biology consistent and reproducible. It is a critical part of the scratch wound assays.
Figure 7. HT-1080 monolayer scratch comparison using an Incucyte® Imagelock 96-Well Plate. Note differences in width uniformity across wells, as well as scratch location within the wells from scratching manually using a 10 µL Pipette tip (top). The Woundmaker Tool accomplishes uniform scratches in all 96 wells with the push of a button (bottom). The time saving is tremendous.
Scratch Wound Assay Technical Resources
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Scratch Wound Frequently Asked Questions
The Incucyte® Scratch Wound Assay measures the movement of cells into a cell-free zone in the absence of a chemotactic gradient. You can use the Incucyte® Woundmaker Tool to create a precise wound in each well and monitor wound closure as cells move either across a substrate (migration) or through a 3D gel matrix (invasion).
The Incucyte® Chemotaxis Assay measures directed cell migration in response to a chemotactic gradient. You can use the Incucyte® Live-Cell Analysis System to investigate treatment effects on chemotactic profile. The system is suitable for adherent and non-adherent cells.
The Incucyte® Imagelock Plate contains fiducial markings designed for the Incucyte® Live-Cell Analysis System to accurately find the same wound region at each image scan. Using the Incucyte® Scratch Wound Analysis Software Module, the system will then normalize wound closure back to the initial time point at each subsequent scan to provide the most accurate and reproducible measurements of cell migration. For this reason, it is highly recommended to use the Incucyte® Imagelock Plate for scratch wound assays.
There are three integrated metrics the Incucyte® Scratch Wound Software Analysis Module uses based on processed images:
Wound Width: The average distance (microns) between the edges of the scratch wound mask
Wound Confluence: The percentage of area of the wound occupied by cells (dependent on the initial scratch wound mask to differentiate wounded from non-wounded area)
Relative Wound Density: Measure of the percentage of density of the wound region relative to the density of the cell region (dependent on the initial scratch wound mask to differentiate wounded from non-wounded area)
Yes. Multiplexed measurements of cell health can be made using any unused imaging channel. The Incucyte® Live-Cell Analysis System supports HD phase-contrast and green, red, orange and near-IR fluorescence automated imaging modes.
Version 2015A Rev 1 or later. The Incucyte® Scratch Wound Analysis Software Module is not compatible with prior versions of software. If you wish to receive a software upgrade, please contact your point-of-sale contact.