The Immunological Synapse. Part B / Clément Thomas and Lorenzo Galluzzi.

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Intro -- The Immunological Synapse -- Part B -- Copyright -- Contents -- Contributors -- Chapter 1: Live-cell imaging for analysis of the NK cell immunological synapse -- 1. Introduction -- 2. Materials -- 2.1. Cells -- 2.2. Culture medium -- 2.3. Reagents -- 2.4. Fluorochromes -- 2.5. Antibodies -- 2.6. Disposables -- 2.7. Equipment -- 2.8. Software -- 3. Methods -- 3.1. Preparation of NK cells and target cells for imaging -- 3.2. Staining and sample preparation -- 3.3. Live-cell imaging -- 3.4. Image editing for presentation -- 3.5. Data analysis and quantification -- 3.5.1. Granule clustering -- 3.5.2. Granule polarization toward the immunological synapse -- 3.5.3. NK cell/cancer cell conjugation, cytotoxicity and IS termination -- Acknowledgments -- References -- Chapter 2: Analyzing the activity of the proteases granzyme B and caspase-8 inside living cells using fluorescence -- 1. Introduction -- 2. Material and methods -- 2.1. Generals and disposables -- 2.2. Equipment -- 2.3. Reagents -- 2.4. HeLa cell culture -- 2.5. Transfection of HeLa cells -- 2.6. Time lapse life cell imaging of fluorescence localization reporter -- 2.7. Data analysis of NES containing reporter constructs -- 3. Concluding remarks -- 4. Notes -- Acknowledgments -- Declaration of interests -- References -- Chapter 3: T cell activation and effector function in the human Jurkat T cell model -- 1. Introduction -- 2. Materials -- 2.1. Common disposables -- 2.2. Common equipment -- 2.3. Analytical software -- 2.4. Cell lines -- 2.5. Reagents -- 2.6. Buffered solutions -- 3. Methods -- 3.1. Maintenance of cell lines -- 3.2. Staphylococcal Enterotoxin E (SEE) dose-response -- 3.2.1. Sample preparation, staining and ELISA -- 3.2.2. Data analysis -- 3.2.2.1. Flow cytometry data -- 3.2.2.2. ELISA data -- 3.3. Synapse assembly -- 3.3.1. Sample preparation and staining.

3.3.2. Sample preparation for actin dynamic analysis by TIRFM -- 3.3.2.1. Cell transfection -- 3.3.2.2. Stimulatory surface preparation -- 3.3.3. Image acquisition -- 3.3.3.1. Confocal microscopy -- 3.3.3.2. TIRFM imaging -- 3.3.4. Image analysis -- 3.3.4.1. Three dimensional (3D) confocal -- 3.3.4.2. TIRFM analysis -- 4. Discussion -- 5. Notes -- Acknowledgments -- References -- Chapter 4: A tractable microscopy- and flow cytometry-based method to measure natural killer cell-mediated -- 1. Introduction -- 2. Protocol -- 2.1. Formation of tumor spheroids -- 2.1.1. Day 0 -- 2.1.2. Day 3-4 -- 2.1.3. Day 6 -- 2.1.3.1. Infiltration, spheroid shrinkage -- 3. Notes -- 4. Concluding remarks -- Acknowledgments -- References -- Chapter 5: Kinetic, imaging based assay to measure NK cell cytotoxicity against adherent cells -- 1. Introduction -- 2. Materials -- 2.1. Disposables -- 2.2. Equipment -- 2.3. Reagents -- 2.4. Cell culture -- 3. Methods -- 3.1. Incucyte Nuclight red-expressing target cell line production -- 3.2. Counting viable NK cells via flow cytometry -- 3.3. Cell culture for kinetic live-cell imaging cytotoxicity assay -- 3.4. Cytotoxicity plate set up -- 3.4.1. Seeding target cells -- For 2D monolayer seeding -- For 3D spheroid seeding -- 3.5. Kinetic live-cell imaging assay -- 3.6. Data analysis -- 3.6.1. Creating an analysis definition -- 3.6.2. Exporting data -- 3.7. Post-live-cell imaging immunological assays -- 4. Concluding remarks -- 5. Notes -- Acknowledgments -- Declaration of interests -- References -- Chapter 6: Correlative light and electron microscopy to explore the lytic immunological synapse between natural killer ce ... -- 1. Introduction -- 2. Materials -- 2.1. Common disposables -- 2.2. Common equipment -- 2.3. Reagents -- 3. Methods -- 3.1. Cell culture -- 3.2. Preparation of effector-target cell conjugates.

3.3. Light microscopy imaging -- 3.4. Sample preparation for electron microscopy -- 3.5. Electron microscopy imaging -- 3.6. Image processing -- 4. Concluding remarks -- 5. Notes -- Acknowledgments -- References -- Chapter 7: Metrics of 2D immunological synapses in human T cells via high-content confocal cell imaging -- 1. Introduction -- 2. Material -- 2.1. Disposables -- 2.2. Equipment -- 2.3. Reagents -- 3. Procedure -- 3.1. T cell culture -- 3.2. Plate coating and sample preparation -- 3.3. T cell stimulation (day 0) -- 3.4. T cell staining (day 0) -- 3.5. Image acquisition -- 3.6. Image analysis -- 4. Concluding remarks -- 5. Notes -- Acknowledgments -- References -- Chapter 8: Kinetic measurements of human CD8+ T cell cytotoxic activity in a 384-well plate format -- 1. Introduction -- 2. Material -- 2.1. Equipment and software -- 2.2. Disposables -- 2.3. Reagents -- 2.4. Cell culture -- 3. Procedure -- 3.1. Coating of the assay plate with fibronectin -- 3.2. Coating of target cells with anti-CD3 antibodies -- 3.3. Staining of cytotoxic T cells -- 3.4. Setting temperature and CO2 conditions in the high-content cell imager -- 3.5. Plate seeding -- 3.6. Image acquisition -- 3.7. Image sequence analysis -- 3.8. Data analysis -- 3.9. Representative results -- 3.10. Notes -- Acknowledgments -- References -- Chapter 9: Imaging PIP2 and BCR microclusters in B cell immunological synapse -- 1. Introduction -- 2. Materials -- 2.1. Common disposables -- 2.2. Chemicals, antibodies, and cells -- 2.2.1. Liposome preparation -- 2.2.2. Chamber preparation -- 2.2.3. Plasmids, antibodies, and cells -- 2.3. Equipment -- 2.4. Software -- 3. Methods -- 3.1. Preparation of PIP2 biosensor -- 3.2. Construction of DT40 cell stably expressing PIP2 biosensor -- 3.3. Liposome preparation -- 3.4. Piranha solution and glass treatment.

3.5. Prepare Alexa Fluor 647-conjugated Fab version antibody -- 3.6. Prepare the imaging chamber with treated glass -- 3.7. PLB containing surrogate antigens preparation -- 3.8. BCR labeling -- 3.9. Time-lapse imaging by TIRFM and data analysis -- 4. Concluding remarks -- Notes -- Acknowledgments -- Disclosure -- References -- Chapter 10: Activation kinetics of regulatory molecules during immunological synapse in T cells -- 1. Introduction -- 2. Materials -- 2.1. Common disposables -- 2.2. Common equipment -- 2.3. Analytical software -- 2.4. Cell lines and primary cells -- 2.5. Reagents -- 2.6. Buffered solutions -- 3. Methods -- 3.1. Isolation of T cells -- 3.1.1. CD4+ T-cell isolation from human peripheral blood lymphocytes (PBLs) -- 3.1.2. Isolation of mouse CD4+ T cells from spleen and lymph nodes -- 3.2. Generation of lymphoblasts from human PBLs: HAp or SEE/SEB-specific -- 3.3. Transfection of T cells -- 3.3.1. Amaxa nucleofection for mouse CD4+ T cells -- 3.3.2. Electroporation for cell lines and human SEE/SEB-specific T lymphoblasts -- 3.4. Signaling assays analyzed by immunoblotting: Stimulating antibodies and cell conjugates -- 3.4.1. Preparation of human SEE/SEB-specific T lymphoblasts or CH7C17 T cells -- 3.4.2. Preload of antigen presenting cells (APCs) with antigen -- 3.4.3. Formation of conjugates -- 3.4.4. Lysis and IB -- 3.5. Stimulation assays analyzed by flow cytometry -- 3.5.1. Cell preparation and staining -- 3.5.2. Flow cytometry data analysis -- 3.6. Imaging of IS formation in conjugates through immunofluorescence analysis -- 3.6.1. Coating of coverslips -- 3.6.2. Formation of conjugates and staining -- 3.6.2.1. Preparation of T cells -- 3.6.2.2. Preparation of APCs -- 3.6.2.3. Formation of conjugates -- 3.6.2.4. Immunofluorescence.

3.7. Imaging of actin dynamics during immune synapse formation in live T cells by confocal microscopy -- 3.7.1. Coating of cell chambers -- 3.7.2. Preparation of T cells -- 3.7.3. Preparation of APCs -- 3.7.4. Formation of conjugates and imaging -- 4. Notes -- Acknowledgments -- Discussion -- References -- Chapter 11: Immunological synapse-driven transfer of extracellular vesicle microRNAs in primary lymphocytes -- 1. Introduction -- 2. Material -- 2.1. Common disposables -- 2.2. Common equipment -- 2.3. Mice -- 2.4. Reagents and buffered solutions -- 2.4.1. Isolation and culture of primary lymphocytes -- 2.4.2. Isolation and culture of primary B lymphocytes -- 2.4.3. Isolation and culture of primary T lymphocytes -- 2.4.4. RNA isolation, real-time PCR and small RNA next-generation sequencing (NGS) -- 2.4.5. Small EV isolation and characterization -- 2.4.6. EV blockade strategies -- 2.4.7. Functional microRNA screening -- 2.5. Analytical software -- 3. Methods -- 3.1. Mice -- 3.2. Cell maintenance and immune synapse co-cultures -- 3.2.1. Isolation and culture of primary T lymphocytes -- 3.2.1.1. Preparation of cell suspension from mouse splenocytes and lymph node cells and culture (Day 0) -- 3.2.1.2. Labeling and isolation of CD4+ T cells (Day 1) -- 3.2.2. Isolation and culture of primary B lymphocytes -- 3.2.2.1. Preparation of cell suspension from mouse splenocytes (Day 0) -- 3.2.2.2. Labeling and isolation of B cells (Day 0) -- 3.2.2.3. Cell culture and activation of B cells (Day 0) -- 3.2.3. Immune synapse co-cultures -- 3.2.4. Strategies for EV release inhibition -- 3.2.4.1. Chemical inhibition -- 3.2.4.2. Genetic inhibition -- 3.2.4.3. Gene knockout mice model -- 3.2.4.4. EV isolation and validation of the inhibition procedures -- 3.3. Immune synapse characterization -- 3.3.1. B and T lymphocytes proliferation.

3.3.2. B and T lymphocytes survival.

This book, 'Methods in Cell Biology: The Immunological Synapse - Part B,' provides an extensive examination of the immunological synapse, focusing on methodologies and techniques applied in cell biology research. Edited by Clement Thomas and Lorenzo Galluzzi, it is a valuable resource for researchers and practitioners in immunology and cell biology. The volume covers a range of topics, including live-cell imaging, analysis of T cell activation, and natural killer cell activity. Aimed at scientists and students in the field, the book offers detailed insights into experimental techniques and their applications in understanding cellular interactions and immune responses. Generated by AI

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