Effect of TNF-α receptor selective mutants on human neutrophil apoptosis. (A) Human neutrophils were incubated with 0.01 to 100 ng/mL of wildtype TNF-α (wtTNF-α, ▪), the TNFR55-selective mutants R32WS86T (•) or E146K (♦), and the TNFR75 selective mutant D143F (▴). Apoptosis was assessed morphologically after 3 hours. Data represent mean ± SEM of three experiments each performed in triplicate. (B) Neutrophils were incubated under identical conditions with 0 to 100 ng/mL wild-type TNF-α, 10 to 100 ng/mL E146K, or 10 to 100 ng/mL D143F. Thereafter the cells were pelletted and incubated overnight in sodium acetate/EDTA/proteinase K/1% SDS before phenol/chloroform DNA extraction as detailed in Materials and Methods. After precipitation with ethanol, 10 μg of DNA was loaded per lane and separated on a 1.2% agarose gel.