Serum inhibits changes in CD16 expression in the absence of viable target cells. (A) Raji cells were fixed in 1% formaldehyde and washed extensively. Fixed cells were mixed with PBMCs at a 1:1 ratio for 20 hours with 5 μg/mL rituximab (n = 3). (B) Flat-bottom plates were coated with 10 μg/mL of rituximab. Plates were washed, and PBMCs were added and cultured for 20 hours. Incubations with fixed Raji cells and rituximab or rituximab-coated plates were preformed in the presence or absence of 50% serum and heat-inactivated serum (n = 5). Surface marker expression was determined using flow cytometry with gating on CD3⁻, CD56⁺ lymphocytes. NK-cell CD16, expressed as median fluorescence, was measured after incubation with media, serum, and heat-inactivated serum in the presence of fixed Raji cells (A) or flat-bottom plates coated with rituximab (B). Error bars represent SD of the mean.