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Selecting the optimal cell migration assay: fundamentals and practical guidelines

Nature Methods volume 23, pages 30–42 (2026)Cite this article

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Abstract

Cell migration is a key cellular process that drives major developmental programs. To mimic and mechanistically understand cell migration in these different contexts, different assays have been developed. However, owing to the lack of practical guidelines, these different cell migration assays are often used interchangeably. This and the inherent dynamic nature of cell migration, which often requires sophisticated live-cell microscopy, may have caused cell migration to be notably less well understood than equally important cell functions, such as cell differentiation or proliferation. In this Review, we describe commonly used custom and commercial in vitro and in vivo cell migration assays and provide a comprehensive practical guide and decision tree outlining how to choose and implement an assay that best suits the biological question at hand. We hope this guidance spurs biological insights into this complex process and encourages future methods development.

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Fig. 1: Commonly used commercial and custom in vitro cell migration assays.
Fig. 2: The wound healing assay.
Fig. 3: The Transwell assay (Boyden chamber assay).
Fig. 4: The 3D cell migration assay.
Fig. 5: The spheroid/organoid invasion assay.

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Acknowledgements

We acknowledge the following sources of support: U54AR081774 (to D.W.), U54CA268083 (to D.W.), R01CA300052 (to D.W.), UG3CA275681 (to P.-H.W.) and UH3CA275681 (to P.-H.W.) all from the National Cancer Institute; and R35-GM157099 (to J.M.P.) from the National Institute of General Medical Sciences and the American Federation for Aging Research Glenn Foundation Junior Faculty Award (to J.M.P.).

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Authors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Wenxuan Du, Andre Forjaz, Jude M. Phillip, Pei-Hsun Wu & Denis Wirtz

  2. Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA

    Wenxuan Du, Praful R. Nair, Andre Forjaz, Jude M. Phillip, Pei-Hsun Wu & Denis Wirtz

  3. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Jude M. Phillip

  4. Translational Therapeutics and Regenerative Engineering Center, Johns Hopkins University, Baltimore, MD, USA

    Jude M. Phillip

  5. Department of Oncology, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, USA

    Jude M. Phillip & Denis Wirtz

  6. Department of Pathology, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, USA

    Denis Wirtz

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Contributions

D.W. conceived the outline. W.D., P.R.N., A.F., J.M.P., P.-H.W. and D.W. wrote the paper. W.D. produced the figures.

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Correspondence to Jude M. Phillip, Pei-Hsun Wu or Denis Wirtz.

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Nature Methods thanks Li Yang, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Du, W., Nair, P.R., Forjaz, A. et al. Selecting the optimal cell migration assay: fundamentals and practical guidelines. Nat Methods 23, 30–42 (2026). https://doi.org/10.1038/s41592-025-02890-1

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