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  • Review Article
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Methods to analyze cell migration data: fundamentals and practical guidelines

Nature Methods volume 23, pages 43–55 (2026)Cite this article

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Abstract

Cell migration assays provide invaluable insights into fundamental biological processes. In a companion Review, we describe commercial and custom in vitro and in vivo assays to measure cell migration and provide guidelines on how to select the most appropriate assay for a given biological question. Here, we describe the fundamental principles of how to compute—from the raw data generated by these assays—quantitative cell migration parameters that help determine the biophysical nature of the cell migration, such as cell speed, mean-squared displacement, diffusivity, persistence, speed and anisotropy, and how to quantify cell heterogeneity, with practical guidance. We also describe new imaging and computational technologies, including AI-based methods, which have helped establish fast, robust and accurate tracking of cells and quantification of cell migration. Taken together, these Reviews offer practical guidance for cell migration assays from conception to analysis.

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Fig. 1: Direct-versus-indirect measurements of cell migration.
Fig. 2: Characterization of cell trajectories and associated metrics.
Fig. 3: MSD for trajectory characterization.
Fig. 4: Indirect analysis of cell migration via common assays.

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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; R35-GM157099 (to J.M.P.) from the National Institute of General Medical Sciences; and an American Federation for Aging Research Glenn Foundation Junior Faculty Award (to J.M.P.).

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Author notes

  1. These authors contributed equally: Pei-Hsun Wu, Jude M. Phillip.

Authors and Affiliations

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

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

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

    Pei-Hsun Wu, Jude M. Phillip, Wenxuan Du, Andre Forjaz, Praful R. Nair & 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., P.-H.W. and J.M.P. conceived the outline. P.-H.W., J.M.P., W.D., A.F., P.R.N. and D.W. wrote the paper.

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Correspondence to Pei-Hsun Wu, Jude M. Phillip 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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Supplementary information

Supplementary Table 1

Mathematical definitions and common metrics used to analyze cell trajectories.

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Wu, PH., Phillip, J.M., Du, W. et al. Methods to analyze cell migration data: fundamentals and practical guidelines. Nat Methods 23, 43–55 (2026). https://doi.org/10.1038/s41592-025-02935-5

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