ADCC of 51Cr-labeled target cells was described previously (52). Briefly, 1 × 106 target cells were labeled with 100 μCi (3.7 MBq) 51Cr for 2 hours. After extensive washing, cells were adjusted to 105/ml. Blood was obtained from healthy donors at the UMC Utrecht. The neutrophil or PMN (polymorphonuclear leukocyte) fraction was isolated from blood by Ficoll/Histopaque separation (GE Healthcare; Sigma-Aldrich). PMNs were pretreated for 15 min at 37°C with TNFα (250 U/ml). Effector cells (40:1 effector-to-target ratio), the monoclonal antibody rituximab at 1 μg/ml, medium, and tumor cells were added to round-bottom microtiter plates (Corning Incorporated). After 4 hours of incubation at 37°C, 51Cr release into the supernatant was assessed by measuring the radioactivity of supernatant samples in counts per minute (cpm). Percentage of specific lysis was calculated using the following formula: [(experimental cpm − basal cpm)/(maximal cpm − basal cpm)] × 100, with maximal lysis determined in the presence of 3% Triton and basal lysis in the absence of antibodies and effector cells.

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