NK cell prestimulation can affect subsequent target cell lysis. (A–D) Primary NK cells were prestimulated with P815, Daudi, Daudi-rituximab (Rtx), or Daudi-MICA. Unstimulated NK cells were used as a control. After 1.5 h incubation, Daudi-rituximab or Daudi-MICA cells labeled with cell trace dye (#) were added and incubated for an additional 1.5 h. (A) Schematic representation of experimental setup. (B) Representative plots of target cell death measured by staining caspase 3/7 activity and assessed by flow cytometry gated on labeled target cells (#). Percentage of caspase 3/7–positive Daudi-rituximab (C) and Daudi-MICA (D). n = 7; mean ± SD; symbols represent different donors. *, P < 0.05; **, P < 0.01 calculated by one-way ANOVA. SSC-W, side scatter–width. See also Fig. S4.