Published: Vol 10, Iss 8, Apr 20, 2020 DOI: 10.21769/BioProtoc.3586 Views: 6259
Reviewed by: Gal HaimovichChaitali BasolePhilipp Wörsdörfer
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
Related protocols
Imaging of Human Cancer Cells in 3D Collagen Matrices
Karin Pfisterer [...] Maddy Parsons
Jan 20, 2021 4924 Views
Studying Chemotactic Migration in Dunn Chamber: An Example Applied to Adherent Cancer Cells
Khedidja Benseddik and Kossay Zaoui
Feb 5, 2022 2063 Views
Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells
Anh H. Le and Laura M. Machesky
Apr 5, 2022 2819 Views
Abstract
Cell migration is a fundamental cellular process that plays a crucial role in many physioglogical and pathological processes such as wound healing or cancer metastasis. Many assays have been developed to examine cell migration, such as the wound healing or scratch assay, Boyden Chamber or transwell assay, and the method we will describe here, single cell migration assay. In this assay, cells are plated sparsely on a collagen coated plate and live cell imaging is performed over a period of 2 h at 1 frame per minute. After imaging is completed, cells are tracked manually using ImageJ by tracking movement of the centroid of the cell. These data points are then exported and overall distance travelled from frame to frame is determined and divided by total time imaged to determine speed of the cell. This method provides a quick way to examine effect of cellular manipulation on cell migration before proceeding to perform more complex assays.
Keywords: Single cell migrationBackground
Cell migration plays an important role in both physiological and pathological processes ranging from embryonic development to angiogenesis and tumor metastasis (Le Clainche and Carlier, 2008). Cell motility is a highly orchestrated event that can be summarized as a cycle of four basic steps: i) membrane protrusion driven by actin polymerization, ii) stabilization of protrusion through integrin-mediated cell-matrix adhesion, iii) cell-body translocation driven by actomyosin contractile force, and finally, iv) rear release as a result of the mechanical action of contractile force and/or proteolysis of cell-matrix adhesion components (Sheetz et al., 1999; Ridley et al., 2003; Panetti et al., 2004; Stradal and Scita, 2006; Tomasevic et al., 2007; Le Clainche and Carlier, 2008). These processes involve dynamic remodeling of actin cytoskeleton which is dependent on de novo synthesis as well as regulation of important structural and regulatory components of actin cytoskeletal system.
Several methods have been developed to examine cell migration and can be separated into 2D and 3D assays. 2D migration assay have their advantages as they are typically easier and quicker to perform, however, lack the physiological representation that 3D assays may provide. We will provide a brief description of some of the other methods for cell migration. The scratch assay or wound healing assay is a commonly used technique to evaluate directed cell migration (Cory, 2011). In this assay, cells are plated to form a confluent monolayer and a stratch is introduced in the monolayer. The cell-free zone is then monitored as adjacent cells migrate to close the gap. While simple to perform, cell proliferation may bias the readout of this assay as that can influence scratch closure. In addition, cells in this assay are forced to move in one direction. The Boyden Chamber assay or transwell assay is another technique used to evaluate cell migration (Falasca et al., 2011). In this assay, cells are placed on one side of a porous membrane and allowed to migrate through the pores to the other side. An advantage of this assay is it allows for chemotaxis and works for both adherent and non-adherent cells. As with the scratch assay, migration in this assay is defined. Some disadvantages of this assay include difficulty to visualize cells and morphology due to transitive state of cells while migrating through pores.
In this protocol, we will describe the use of single cell migration assay to examine MDA-MB-231 cell migration. The key difference in this protocol is that we examine random cell migration rather than directed migration. This allows us to examine cell migration in a manner that is less influenced by cell-cell contact and its innate ability for directional persistence, i.e., ability to migrate randomly in a single direction. While obviously not a true physiological representation of in vivo cell migration, this assay allows us to quickly analyze effect of cellular manipulation on its ability to migrate. While this assay uses MDA-MB-231, this protocol can be performed with any migratory cell line. We have performed this assay using multiple cell lines with minor modifications (media used and amount of cells plated).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
This work was supported by a grant from the National Institute of Health (2R01CA108607) to PR. David Gau was supported by a National Science Foundation pre-doctoral fellowship (2012139050) and an NIH Cardiovascular Bioengineering pre-doctoral training grant (2T32HL076124 to SG).
Competing interests
The authors have no competing interests to report.
References
Article Information
Copyright
© 2020 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Category
Cancer Biology > Invasion & metastasis > Cell biology assays
Cell Biology > Cell movement > Cell migration
Cell Biology > Cell imaging > Live-cell imaging
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link