Abstract
Cell migration is a highly complex and dynamic biological process, essential in several physiological phenomena and pathologies including cancer dissemination and metastasis formation. Thus understanding single cell migration is highly relevant and requires a suitable image-based assay. Depending on the speed of the moving cells, one may require fast time-lapse microscopy, which is not always suitable for high-throughput screening. To overcome this, a quantitative and fixed single cell migration assay was developed based on the PhagoKinetic Tracks (PKT) procedure. Briefly, cells are seeded on top of a monolayer of carboxylated latex beads, and as cells migrate, they phagocytose these beads and leave behind a migratory track. These bead-free migratory tracks can be visualized using a standard bright field microscope and analysed for a multiparametric quantitative assessment of single cell migration (Naffar-Abu-Amara et al., 2008).Here we describe a detailed and optimized protocol of the PKT assay, adaptable for both RNAi and drug screening (van Roosmalen et al., 2015). This protocol allows the user to study migratory behaviour at the single cell level, without fast and live-imaging microscopy.
Keywords: Cell migration, Microscopy, High throughput, Screening, Image analysis
Materials and Reagents
Equipment
Software
Procedure
Notes
Important considerations
Recipes
Acknowledgments
This protocol was developed based on Naffar-Abu-Amara et al. (2008) and further optimized in the Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University. The work was funded by grants from the EU-FP7 - Systems Microscopy NoE (grant no. 258068 to B. van de Water) and the Dutch Cancer Society (UL2007-3860).
References
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