The Immune Synapse Methods and Protocols

This new collection features the most up-to-date essential protocols that are currently being used to study the immune synapse. Beginning with methods for making biophysical measurements, the volume continues by covering the cell biology of synapses, methods for advanced substrate engineering, mechanobiology topics, new technologies to describe and manipulate synaptic components, as well as methods related to sites of action and immunotherapy. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Measuring the Co-Localization and Dynamics of Mobile Proteins in Live Cells Undergoing Signaling Responses.- Fluorescence-Based Measurements of Two-Dimensional Affinity in Membrane Interfaces.- Surfaces for Study of Receptor Dynamics on T Cells.- Molecular Dynamics Simulations of Immune Receptors and Ligands.- Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS).- Combined FRET-FLIM and NAD(P)H FLIM to Analyze B Cell Receptor Signaling Induced Metabolic Activity of Germinal Center B Cells In Vivo.- Measurement of Molecular Height Using Cell Surface Optical Profilometry (CSOP).- Observing Membrane and Cell Adhesion via Reflection Interference Contrast Microscopy (RICM).- En-Face-Imaging of T Cell-Dendritic Cell Immunological Synapses.- High and Super Resolution Imaging of Cell-Cell Interfaces.- Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immuno-Isolation.- Microvillar Cartography: A Super-Resolution Single-Molecule Imaging Method to Map the Positions of Membrane Proteins with Respect to Cellular Surface Topography.- T Cell Immunological Synaptosomes: Definition and Isolation.- Dynamics of Immune Cell Microvilli.- Visualization of Myddosome Assembly in Live Cells.- Detection of Telomere Transfer at Immunological Synapse.- Bottom-Up Assembly of Bio-Inspired, Fully Synthetic Extracellular Vesicles.- A DNA Origami-Based Biointerface to Interrogate the Spatial Requirements for Sensitized T-Cell Antigen Recognition.- Leveraging DNA Origami to Study Phagocytosis.- Fabrication of Nanoscale Arrays to Study the Effect of Ligand Arrangement on Inhibitory Signaling in NK Cells.- Measurement of Forces for Trans-Endocytosis at Dorsal and Ventral Sides of the Cell.- Functionalized Lipid Droplets and Microfluidics Approach to Study Immune Cell Polarity In Vitro.- Quantifying Immune Cell Force Generation Using Traction Force Microscopy.- Characterizing Biophysical Parameters of Single TCR-pMHC Interactions Using Optical Tweezers.- Isolation of the B Cell Immune Synapse for Proteomic Analysis.- Analyzing Single Cell Secretions by ‘Shadow Imaging’.- Exploiting the RUSH System to Study Lytic Granule Biogenesis in Cytotoxic T Lymphocytes.- Interactions of Tissue Resident T Cells.- Live Imaging of CAR T Cell Ca²⁺ Signals in Tumor Slices Using Confocal Microscopy.- Measuring CTL Lytic Granule Secretion and Target Cell Membrane Repair by Fluorescent Lipophilic Dye Uptake at the Lytic Synapse.- In Vitro Generation of Human Tolerogenic Monocyte-Derived Dendritic Cells.- Methods of Machine Learning-Based Chimeric Antigen Receptor Immunological Synapse Quality Quantification.- Imaging CAR-T Synapse as a Quality Control for CAR Engineering.