Immune cells are constantly on the move through multicellular organisms to explore and respond to pathogens and other harmful insults. While moving, immune cells efficiently traverse microenvironments composed of tissue cells and extracellular fibers, which together form complex environments of various porosity, stiffness, topography, and chemical composition. In this protocol we describe experimental procedures to investigate immune cell migration through microenvironments of heterogeneous porosity. In particular, we describe micro-channels, micro-pillars, and collagen networks as cell migration paths with alternative pore size choices. Employing micro-channels or micro-pillars that divide at junctions into alternative paths with initially differentially sized pores allows us to precisely (1) measure the cellular translocation time through these porous path junctions, (2) quantify the cellular preference for individual pore sizes, and (3) image cellular components like the nucleus and the cytoskeleton. This reductionistic experimental setup thus can elucidate how immune cells perform decisions in complex microenvironments of various porosity like the interstitium. The setup further allows investigation of the underlying forces of cellular squeezing and the consequences of cellular deformation on the integrity of the cell and its organelles. As a complementary approach that does not require any micro-engineering expertise, we describe the usage of three-dimensional collagen networks with different pore sizes. Whereas we here focus on dendritic cells as a model for motile immune cells, the described protocols are versatile as they are also applicable for other immune cell types like neutrophils and non-immune cell types such as mesenchymal and cancer cells. In summary, we here describe protocols to identify the mechanisms and principles of cellular probing, decision making, and squeezing during cellular movement through microenvironments of heterogeneous porosity.
We thank Kasia Stefanowski for excellent technical assistance, and the Core Facility Bioimaging of the Biomedical Center (BMC) of the Ludwig-Maximilian University for excellent support. We gratefully acknowledge financial support from the Peter Hans Hofschneider Professorship of the Stiftung Experimentelle Biomedizin (to J.R), from the DFG (Collaborative Research Center SFB914, project A12; and Priority Programme SPP2332, project 492014049; both to J.R) and from the LMU Institutional Strategy LMU-Excellent within the framework of the German Excellence Initiative (to J.R). Open access funding enabled and organized by Projekt DEAL.
Kroll J, Ruiz-Fernandez MJA, Braun MB, Merrin J, Renkawitz J. Quantifying the probing and selection of microenvironmental pores by motile immune cells. Current Protocols. 2022;2(4). doi:10.1002/cpz1.407
Kroll, J., Ruiz-Fernandez, M. J. A., Braun, M. B., Merrin, J., & Renkawitz, J. (2022). Quantifying the probing and selection of microenvironmental pores by motile immune cells. Current Protocols. Wiley. https://doi.org/10.1002/cpz1.407
Kroll, Janina, Mauricio J.A. Ruiz-Fernandez, Malte B. Braun, Jack Merrin, and Jörg Renkawitz. “Quantifying the Probing and Selection of Microenvironmental Pores by Motile Immune Cells.” Current Protocols. Wiley, 2022. https://doi.org/10.1002/cpz1.407.
J. Kroll, M. J. A. Ruiz-Fernandez, M. B. Braun, J. Merrin, and J. Renkawitz, “Quantifying the probing and selection of microenvironmental pores by motile immune cells,” Current Protocols, vol. 2, no. 4. Wiley, 2022.
Kroll J, Ruiz-Fernandez MJA, Braun MB, Merrin J, Renkawitz J. 2022. Quantifying the probing and selection of microenvironmental pores by motile immune cells. Current Protocols. 2(4), e407.
Kroll, Janina, et al. “Quantifying the Probing and Selection of Microenvironmental Pores by Motile Immune Cells.” Current Protocols, vol. 2, no. 4, e407, Wiley, 2022, doi:10.1002/cpz1.407.
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