Numbers matter: quantitative and dynamic analysis of the formation of a mature immunological synapse using imaging flow cytometry (42.15)

Abstract

Abstract Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following intercellular contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. We use multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, to visualize and quantify the recruitment of signaling molecules CD3ε and Lck during the evolution of a mature immune synapse. We use two independent analysis methods to show that the dynamics of Lck and CD3ε recruitment are comparable; however, CD3ε exhibits longer residence times (> 8 min) following Ca++ triggered activation in the synapses of thousands of T cell-macrophage conjugates.