Live Cell Imaging and Analysis to Capture T-Cell Motility in Real-Time

  • Seow Theng OngEmail author
  • Navin Kumar Verma
Part of the Methods in Molecular Biology book series (MIMB, volume 1930)


T-lymphocytes are the principle coordinators of the immune defense system and play a major role in the protection of our body against infections, intruders of non-self, and malignancies. To mount an immune response, T-cells need to be effectively employed to tissue sites of infection or inflammation and establish contacts with antigen-presenting cells (APCs) or malignant cells. Understanding how T-cells navigate toward their recruitment sites would offer new therapeutic opportunities. Advancement in the hardware and software upgrades of microscopy technology has created several efficient and easy-to-operate live cell imaging platforms. In this protocol, we present a generalized and simple-to-follow protocol for live cell imaging of migrating T-cells, which can also be adopted to visualize real-time tracking of intracellular signaling events.

Key words

Live cell imaging Advanced microscopy T-cell migration Cell tracking 



This work was supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant to N.K.V. and the Singapore Ministry of Education (MOE) under its Singapore MOE Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004). The authors would like to thank Jaron Liu (GE Healthcare) and Steve Chai (Bitplane AG) for their technical advice on the functionality of the equipment and software.


  1. 1.
    Esner M, Meyenhofer F, Bickle M (2018) Live-cell high content screening in drug development. Methods Mol Biol 1683:149–164CrossRefGoogle Scholar
  2. 2.
    Ong ST, Chalasani MLS, Fazil MHUT, Prasannan P, Kizhakeyil A, Wright GD, Kelleher D, Verma NK (2018) Centrosome- and Golgi-localized protein kinase N-associated protein serves as a docking platform for protein kinase A signaling and microtubule nucleation in migrating T-cells. Front Immunol 9:397CrossRefGoogle Scholar
  3. 3.
    Buchser W, Collins M, Garyantes T et al (2014) Assay development guidelines for image-based high content screening, high content analysis and high content imaging. In: Assay guidance manual [Internet]. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda, MD, p 2004–2012Google Scholar
  4. 4.
    Ong ST, Freeley M, Skubis-Zegadło J, Fazil MHUT, Kelleher D, Fresser F, Baier G, Verma NK, Long A (2014) Phosphorylation of Rab5a protein by protein kinase Cϵ is crucial for T-cell migration. J Biol Chem 289:19420–19434CrossRefGoogle Scholar
  5. 5.
    Dixit R, Cyr R (2003) Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J 36:280–290CrossRefGoogle Scholar
  6. 6.
    Hoebe RA, Van Oven CH, Gadella TWJ, Dhonukshe PB, Van Noorden CJF, Manders EMM (2007) Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nat Biotechnol 25:249–253CrossRefGoogle Scholar
  7. 7.
    Bernas T, ZarE¸Bski M, Cook RR, Dobrucki JW (2004) Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux. J Microsc 215:281–296CrossRefGoogle Scholar
  8. 8.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore
  2. 2.Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore

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