Surface organization of CD20 influences efficiency of target cell killing. Daudi/CD20-GFP cells were preincubated with 10 µg/mL rituximab and then mixed with freshly isolated primary NK cells and imaged for 40 to 60 minutes. (A) A schematic representation (left panel) and 3D reconstructed snapshots from live cell microscopy (right panels) of Daudi cells with unpolarized CD20 interacting with and being killed by an NK cell (CD20 is green, LysoTracker used to visualize lytic granules within NK cells is red, Sytox blue used to visualize dead cells is white). (B) A schematic representation (left) and 3-dimensional reconstructed snapshots from live cell microscopy (right) show examples of target cell killing in the context of CD20 organization when CD20 is capped: cells with CD20 capping away from the site of contact with an NK cell (top panels) and cells with CD20 enriched on the side of the initial contact with an NK cell (bottom panels). Scale bars represent 10 μm. (C) Proportion of conjugates with or without capping of CD20 in which killing of a target cell took place. (D) Proportion of conjugates with capping of CD20 away from the initial contact or at the contact side in which killing of a target cell took place. (E) Time of conjugation between Daudi and NK cell leading to killing of the target was quantified for each category as well as for Daudi-MICA in conjugates with NK cells.