The Immunological Synapse - Part C, Volume 193 in the Methods in Cell Biology series provides state-of-the-art and detailed methods for the study of the T cell, natural killer (NK) cell and B cell immunological synapses. Topics covered in this third volume include tau-STED microscopy for 3D-quantitative colocalization of lytic granule markers, image processing approaches for 3D-analysis of markers and for microtubule remodeling quantification at the immunological synapse, flow cytometry to analyze NK cell-target cell conjugation, imaging flow cytometry to quantify a range of different subcellular processes at the immunological synapse, imaging techniques aimed at quantitatively analyzing B cell immunological synapse formation, quantification of force-mediated antigen extraction in the B cell immune synapse using DNA-based tension sensors, measuring interaction forces between T lymphocytes and their target cells using live microscopy and laminar shear flow chambers, measuring interaction kinetics between T cells and their target tumor cells with optical tweezers, and gauging antigen recognition by human primary T-cells featuring orthotopically exchanged TCRs of choice. Additionally, this volume includes methods to isolate and characterize primary NK cells and enrich the KIR2DL1+ population, and to expand ?d T cells and evaluate their cytotoxicity.