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A ring barrier–based migration assay to assess cell migration in vitro

Nature Protocols volume 10, pages 904–915 (2015)Cite this article

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Abstract

Cell migration is a key feature of virtually every biological process, and it can be studied in a variety of ways. Here we outline a protocol for the in vitro study of cell migration using a ring barrier–based assay. A 'barrier' is inserted in the culture chamber, which prevents cells from entering a defined area. Cells of interest are seeded around this barrier, and after the formation of a peripheral monolayer the barrier is removed and migration into the cell-free area is monitored. This assay is highly reproducible and convenient to perform, and it allows the deduction of several parameters of migration, including total and effective migration, velocity and cell polarization. An advantage of this assay over the conventional scratch assay is that the cells move over an unaltered and virgin surface, and thus the effect of matrix components on cell migration can be studied. In addition, the cells are not harmed at the onset of the assay. Through computer automation, four individual barrier assays can be monitored at the same time. The procedure can be used in a 12-well standard plate allowing higher throughput, or it can be modified to perform invasion assays. The basic procedure takes 2–3 d to complete.

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Figure 1: Ring barrier–based migration assay.
Figure 2: Ring barrier configurations.
Figure 3: Flow diagram of the general protocol with the time course, the main steps in solid boxes and the options in dashed boxes.
Figure 4: Cell migration unit and configuration.
Figure 5: Migratory parameters.
Figure 6: Migratory responses to ECM coatings.
Figure 7: Co-culture assay and invasion assay.
Figure 8: Immunofluorescence stainings.

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Acknowledgements

We thank A. Brouwer and colleagues of the Erasmus Medical Instrumentation Service for assistance with the development of the migration barriers.

Author information

Authors and Affiliations

  1. Department of Surgery, Laboratory of Experimental Surgical Oncology, Section Surgical Oncology, Erasmus Medical Center, Rotterdam, the Netherlands

    Asha M Das, Alexander M M Eggermont & Timo L M ten Hagen

  2. Institut de Cancérologie Gustave Roussy, Villejuif, France

    Alexander M M Eggermont

Authors
  1. Asha M Das
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  2. Alexander M M Eggermont
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  3. Timo L M ten Hagen
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Contributions

A.M.D. and T.L.M.t.H. conceived the project. A.M.D. performed experiments and data analysis. A.M.D. and T.L.M.t.H. prepared the figures. A.M.D. wrote the manuscript and A.M.M.E. and T.L.M.t.H. critically revised it.

Corresponding author

Correspondence to Timo L M ten Hagen.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cell migration barrier.

The cell migration barrier consists of a surgical stainless steel cylinder and a three legged spacer made of polyether ether ketone (PEEK). The basic dimensions of the cell migration barrier are provided.

Supplementary Figure 2 Cell invasion barrier.

The cell invasion barrier consists of a surgical stainless steel cylinder and a three legged spacer made of polyether ether ketone (PEEK). The basic dimensions of the cell invasion barrier are provided.

Supplementary Figure 3 Cell migration unit configurations.

Images of cell migration unit configurations for imaging (a) multiple cell chambers (b) single cell chamber and (c) 12-well plates with the unit components: Cell migration units (1), Cell migration heater (2), Anti-fog glass heater (3), Gas humidifier/heater (4) and suitable microscopes (5).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 122 kb)

Supplementary Manual 1

Interactive PDF of the cell migration barrier. This interactive file can be viewed using Adobe Acrobat XI or Adobe Acrobat Pro. The migration barrier can be viewed in 3D and dimensions measured using the ‘3D measurement tool’ under the ‘Tools’ menu. To access the ‘Tools’ menu, right click on the figure. Scaling, 1 model unit = 1000 mm. (PDF 508 kb)

Supplementary Manual 2

Interactive PDF of the cell invasion barrier. This interactive file can be viewed using Adobe Acrobat XI or Adobe Acrobat Pro. The invasion barrier can be viewed in 3D and dimensions measured using the ‘3D measurement tool’ under the ‘Tools’ menu. To access the ‘Tools’ menu, right click on the figure. Scaling, 1 model unit= 1000 mm. (PDF 500 kb)

Supplementary Data 1

STEP file for migration barrier. (TXT 149 kb)

Supplementary Data 2

IGS file for migration barrier. (TXT 398 kb)

Supplementary Data 3

STEP file for invasion barrier. (TXT 98 kb)

Supplementary Data 4

IGS file for invasion barrier. (TXT 266 kb)

Ring-barrier based migration assay. (MOV 29491 kb)

Metastatic melanoma (MM) cell migration on fibronectin coating.

Cell migration was imaged by time-lapse phase-contrast microscopy and an image was collected every 12 min for 16 h. The movie is set to a display rate of 10 frames/s and takes 8s. Original magnification ×10. (MOV 5280 kb)

Non-metastatic melanoma (NM) cell migration on fibronectin coating.

Cell migration was imaged by time-lapse phase-contrast microscopy and an image was collected every 12 min for 16 h. The movie is set to a display rate of 10 frames/s and takes 8s. Original magnification ×10. (MOV 3645 kb)

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Das, A., Eggermont, A. & ten Hagen, T. A ring barrier–based migration assay to assess cell migration in vitro. Nat Protoc 10, 904–915 (2015). https://doi.org/10.1038/nprot.2015.056

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