TNFα does not contribute to PMN-SA lysis. (A) HT-29 cells were pretreated without or with varied concentrations of TNFα neutralizing antibody (D1B4) and then stimulated with CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using repeated measures 1-way ANOVA and Tukey posttest (*P < .05 vs untreated, ***P < .0001 vs untreated, and ^#P < .05 vs CZT + 0, 10, or 100 ng/ml D1B4). (B) PMN were pretreated without or with varied concentrations of D1B4 and then incubated without or with SA. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (***P < .0001 vs SA). (C) Reduced caspase 8 immunoprecipitates from sonicates of PMN or PMN-SA at 0, 1, and 3 hours after phagocytosis were probed for the presence of FADD, RIPK-3, RIPK-1, and caspase 8 by immunoblotting. Shown is a representative experiment of 5.