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This volume discusses the latest developments in cellular, molecular, biochemical, and imaging assays to study the biology and functions of T-cells. The chapters in this book cover topics such as LFA-1/ICAM-1 interactions in T-cell motility; using 3D-SIM to dissect signaling cross-talks in motile T-cells; GapmeR-mediated gene silencing in motile T-cells; activity of cellular kinases in migrating T-cells; and computational analysis of protein-protein interactions in motile T-cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and comprehensive, T-Cell Motility: Methods and Protocols is an essential resource for graduate students, postdoctoral fellows, and principal investigators working in the fields of immunology, T-cell biology, biochemistry, molecular biology, and imaging.

T-Cell Motility: Methods and Protocols

Preface...

Table of Contents...

Contributing Authors...



1. An Introduction to LFA-1/ICAM-1 Interactions in T-Cell Motility

Navin Kumar Verma and Dermot Kelleher



2. Isolation of Human Peripheral Blood T Lymphocytes

Atish Kizhakeyil, Seow Theng Ong, Mobashar Hussain Urf Turabe Fazil, Madhavi Latha Somaraju Chalasani, Praseetha Prasannan, and Navin Kumar Verma



3. A Laboratory Model to Study T-Cell Motility

Navin Kumar Verma, Sjyam Mohan Palapetta, Seow Theng Ong, Mobashar Hussain Urf Turabe Fazil, Madhavi Latha Somaraju Chalasani, Praseetha Prasannan, Atish Kizhakeyil, and Dermot Kelleher



4. Quantification of T-Cell Migratory Phenotypes using High Content Analysis

Aik Seng Ng, Seow Theng Ong, Dermot Kelleher, and Navin Kumar Verma



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

Seow Theng Ong and Navin Kumar Verma



6. Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signalling Cross-Talks in Motile T-Cells

Seow Theng Ong, Graham D. Wright, and Navin Kumar Verma



7. Real-Time Impedance-Based Detection of LFA-1-Stimulated T-Cell Transwell Chemotaxis

Praseetha Prasannan and Navin Kumar Verma



8. Quantitative Real-Time PCR for Evaluating Transcriptional Changes in T-Lymphocytes

Atish Kizhakeyil and Navin Kumar Verma



9. GapmeR-Mediated Gene Silencing in Motile T-Cells

Mobashar Hussain Urf Turabe Fazil, Seow Theng Ong, Madhavi Latha Chalasani, Atish Kizhakeyil, and Navin Kumar Verma



10. Live Imaging of Resident T-Cell Migration in Human Lymphoid Tissue Slices using Confocal Microscopy

Emmanuel Donnadieu, Yvonne Michel, and Martin-Leo Hansmann



11. Phosphoprotein Enrichment for Protein Analysis in Motile T-Lymphocytes

Praseetha Prasannan, Rathi Saravanan, and Navin Kumar Verma



12. Immunometabolomic Phenotyping of Motile T-Cells

Madhavi Latha Somaraju Chalasani, Gaelin Looi Wen Dong, and Navin Kumar Verma



13. Profiling Activity of Cellular Kinases in Migrating T-Cells

Chandra Sekhar Chirumamilla, Mobashar Hussain Urf Turabe Fazil, Claudina Perez-Novo, Savithri Rangarajan, Rik de Wijn, Padma Ramireddy, Navin Kumar Verma, and Wim Vanden Berghe



14. Biotinylation Assay to Determine LFA-1 Recycling in Motile T-Lymphocytes

Malin Samuelsson and Lena M. Svensson



15. A Protocol to Study T-Cell Signalling in an Immune Synapse by Microscopy

Mobashar Hussain Urf Turabe Fazil, Pankal Kumar, and Navin Kumar Verma



16. Enzyme-Linked Immunosorbent Assay for T-Cell Dependent Immunogenicity Assessment of Therapeutic Peptides

Madhavi Latha Somaraju Chalasani, Rajamani Lakshminarayanan, and Navin Kumar Verma



17. Modified Intravital Microscopy to Assess Vascular Health and T-Cell Motility

Geoffrey W. Payne, Kevin Mitchell, and Stephanie L. Sellers



18. Computational Analysis of Protein-Protein Interactions in Motile T-Cells

Sunil Kumar, Mobashar Hussain Urf Turabe Fazil, Khursheed Ahmad, Minaketan Tripathy, Jagath Chandana Rajapakse, and Navin Kumar Verma