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