, Volume 51, Issue 1, pp 7–19

Automated time-lapse microscopy and high-resolution tracking of cell migration

  • Joseph S. Fotos
  • Vivek P. Patel
  • Norman J. Karin
  • Murali K. Temburni
  • John T. Koh
  • Deni S. Galileo
Original Paper


We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in “scratch” or “wound healing” assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.


Cell migration Green fluorescent protein Scratch assay Time-lapse Tumor cell lines Vital fluorescent labeling 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Besson A, Gurian-West M, Schmidt A, Hall A, Roberts JM (2004) p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 18:862–876CrossRefGoogle Scholar
  2. Cretu A, Fotos JS, Little BW, Galileo DS (2005) Human and rat glioma growth, invasion, and vascularization in a novel chick embryo brain tumor model. Clin Exper Metas 22:225–236CrossRefGoogle Scholar
  3. Edme N, Downward J, Thiery JP, Boyer B (2002) Ras induces NBT-II epithelial cell scattering through the coordinate activities of Rac and MAPK pathways. J Cell Sci 115:2591–2601Google Scholar
  4. Endo Y, Wolf V, Muraiso K, Kamijo K, Soon L, Uren A, Barshishat-Kupper M, Rubin JS (2005) Wnt-3a-dependent cell motility involves RhoA activation and is specifically regulated by dishevelled-2. J Biol Chem 280:777–786Google Scholar
  5. Galileo DS, Gee AP, Linser PJ (1991) Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion. Dev Biol 146:278–291CrossRefGoogle Scholar
  6. Gavert N, Conacci-Sorrell M, Gast D, Schneider A, Altevogt P, Brabletz T, Ben-Ze’ev A (2005) L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers. J Cell Biol 168:633–642CrossRefGoogle Scholar
  7. Harms BD, Bassi GM, Horwitz AR, Lauffenburger DA (2005) Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions. Biophys J 88:1479–1488CrossRefGoogle Scholar
  8. Herren B, Garton KJ, Coats S, Bowen-Pope DF, Ross R, Raines EW (2001) ADAM15 overexpression in NIH3T3 cells enhances cell–cell interactions. Exp Cell Res 271:152–160CrossRefGoogle Scholar
  9. Hoang MV, Whelan MC, Senger DR (2004) Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Proc Natl Acad Sci USA 101:1874–1879CrossRefGoogle Scholar
  10. Huang C, Rajfur Z, Borchers C, Schaller MD, Jacobson K (2003) JNK phosphorylates paxillin and regulates cell migration. Nature 424:219–223CrossRefGoogle Scholar
  11. John GR, Chen L, Rivieccio MA, Melendez-Vasquez CV, Hartley A, Brosnan CF (2004) Interleukin-1beta induces a reactive astroglial phenotype via deactivation of the Rho GTPase-Rock axis. J Neurosci 24:2837–2845CrossRefGoogle Scholar
  12. Laurent-Matha V, Maruani-Herrmann S, Prebois C, Beaujouin M, Glondu M, Noel A, Alvarez-Gonzalez ML, Blacher S et al (2005) Catalytically inactive human cathepsin D triggers fibroblast invasive growth. J Cell Biol 168:489–499CrossRefGoogle Scholar
  13. Lee CC, Putnam AJ, Miranti CK, Gustafson M, Wang LM, Vande Woude GF, Gao CF (2004) Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene 23:5193–5202CrossRefGoogle Scholar
  14. Lynch L, Vodyanik PI, Boettiger D, Guvakova MA (2005) Insulin-like growth factor I controls adhesion strength mediated by alpha5beta1 integrins in motile carcinoma cells. Mol Biol Cell 16:51–63CrossRefGoogle Scholar
  15. Maschler S, Wirl G, Spring H, Bredow DV, Sordat I, Beug H, Reichmann E (2005) Tumor cell invasiveness correlates with changes in integrin expression and localization. Oncogene 24:2032–2041CrossRefGoogle Scholar
  16. Miao H, Strebhardt K, Pasquale EB, Shen TL, Guan JL, Wang B (2005) Inhibition of integrin-mediated cell adhesion but not directional cell migration requires catalytic activity of EphB3 receptor tyrosine kinase. Role of Rho family small GTPases. J Biol Chem 280:923–932CrossRefGoogle Scholar
  17. Motegi S, Okazawa H, Ohnishi H, Sato R, Kaneko Y, Kobayashi H, Tomizawa K, Ito T et al (2003) Role of the CD47-SHPS-1 system in regulation of cell migration. EMBO J 22:2634–2644CrossRefGoogle Scholar
  18. Moyano JV, Maqueda A, Casanova B, Garcia-Pardo A (2003) Alpha4beta1 integrin/ligand interaction inhibits alpha5beta1-induced stress fibers and focal adhesions via down-regulation of RhoA and induces melanoma cell migration. Mol Biol Cell 14:3699–3715CrossRefGoogle Scholar
  19. Nishio T, Kawaguchi S, Yamamoto M, Iseda T, Kawasaki T, Hase T (2005) Tenascin-C regulates proliferation and migration of cultured astrocytes in a scratch wound assay. Neuroscience 132:87–102CrossRefGoogle Scholar
  20. Petridis AK, El-Maarouf A, Rutishauser U (2004) Polysialic acid regulates cell contact dependent neuronal differentiation of progenitor cells from the subventricular zone. Dev Dyn 230:675–684CrossRefGoogle Scholar
  21. Piccolo E, Innominato PF, Mariggio MA, Maffucci T, Iacobelli S, Falasca M (2002) The mechanism involved in the regulation of phospholipase Cgamma1 activity in cell migration. Oncogene 21:6520–6529CrossRefGoogle Scholar
  22. Pratt SJ, Epple H, Ward M, Feng Y, Braga VM, Longmore GD (2005) The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration. J Cell Biol 168:813–824CrossRefGoogle Scholar
  23. Raftopoulou M, Etienne-Manneville S, Self A, Nicholls S, Hall A (2004) Regulation of cell migration by the C2 domain of the tumor suppressor PTEN. Science 303:1179–1181CrossRefGoogle Scholar
  24. Ray RM, McCormack SA, Covington C, Viar MJ, Zheng Y, Johnson LR (2003) The requirement for polyamines for intestinal epithelial cell migration is mediated through Rac1. J Biol Chem 278:13039–13046CrossRefGoogle Scholar
  25. Robinet A, Fahem A, Cauchard JH, Huet E, Vincent L, Lorimier S, Antonicelli F, Soria C et al (2005) Elastin-derived peptides enhance angiogenesis by promoting endothelial cell migration and tubulogenesis through upregulation of MT1-MMP. J Cell Sci 118:343–356CrossRefGoogle Scholar
  26. Six EM, Ndiaye D, Sauer G, Laabi Y, Athman R, Cumano A, Brou C, Israel A, Logeat F (2004) The notch ligand Delta1 recruits Dlg1 at cell–cell contacts and regulates cell migration. J Biol Chem 279:55818–55826CrossRefGoogle Scholar
  27. Sun S, Wise J, Cho M (2004) Human fibroblast migration in three-dimensional collagen gel in response to noninvasive electrical stimulus. I. Characterization of induced three-dimensional cell movement. Tissue Eng 10:1548–1557Google Scholar
  28. Wadham C, Gamble JR, Vadas MA, Khew-Goodall Y (2003) The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin. Mol Biol Cell 14:2520–2529CrossRefGoogle Scholar
  29. Yarrow JC, Perlman ZE, Westwood NJ, Mitchison TJ (2004) A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol 4:21CrossRefGoogle Scholar
  30. Yoshida H, Cheng W, Hung J, Montell D, Geisbrecht E, Rosen D, Liu J, Naora H (2004) Lessons from border cell migration in the Drosophila ovary: a role for myosin VI in dissemination of human ovarian cancer. Proc Natl Acad Sci USA 101:8144–8149CrossRefGoogle Scholar
  31. Zhang L, Deng M, Parthasarathy R, Wang L, Mongan M, Molkentin JD, Zheng Y, Xia Y (2005) MEKK1 transduces activin signals in keratinocytes to induce actin stress fiber formation and migration. Mol Cell Biol 25:60–65CrossRefGoogle Scholar
  32. Zhu N, Lalla R, Eves P, Brown TH, King A, Kemp EH, Haycock JW, MacNeil S (2004) Melanoma cell migration is upregulated by tumour necrosis factor-alpha and suppressed by alpha-melanocyte-stimulating hormone. Brit J Cancer 90:1457–1463CrossRefGoogle Scholar
  33. Zhu X, Jiang J, Shen H, Wang H, Zong H, Li Z, Yang Y, Niu Z et al (2005) Elevated beta1,4-galactosyltransferase I in highly metastatic human lung cancer cells. Identification of E1AF as important transcription activator. J Biol Chem 280:12503–12516CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Joseph S. Fotos
    • 1
  • Vivek P. Patel
    • 1
  • Norman J. Karin
    • 1
    • 3
  • Murali K. Temburni
    • 1
    • 2
  • John T. Koh
    • 2
  • Deni S. Galileo
    • 1
  1. 1.Department of Biological SciencesUniversity of DelawareNewarkUSA
  2. 2.Chemistry and BiochemistryUniversity of DelawareNewarkUSA
  3. 3.Pacific Northwest National LaboratoryRichlandUSA

Personalised recommendations