Necrostatin 1 nec 1

DCs were seeded in 96-well round-bottom plates at 1 × 10⁵ cells per well in a final volume of 100 µl of RPMI medium without phenol red (Gibco) that was supplemented with 0.05% human serum albumin (Seracare) and 10 mM HEPES. DCs were infected with S. aureus at a multiplicity of infection (MOI) of 25, 10, 5, 1, or 0.1 and incubated at 37°C under shaking conditions at 180 rpm for 15 min, 1 h, 2 h, or 4 h. To assess the cell death pathways, DCs were incubated with one of the inhibitors necrostatin-1 (Nec-1; Enzo Life Sciences), necrosulfonamide (NSA; Calbiochem), GSK-872 (Calbiochem), VX-765 (Calbiochem), Ac-DEVD-CHO (Enzo Life Sciences), or Z-VAD-FMK (Selleck Chemicals) for 30 min at 37˚C and 5% CO₂ without shaking prior to infection with S. aureus. Following infection, cells were pelleted by centrifugation at 1,500 rpm at 4°C for 5 min and lactate dehydrogenase (LDH) release was measured using the CytoTox-ONE homogeneous membrane integrity assay (Promega). In brief, 25 µl of culture supernatant was mixed with 25 µl of LDH reagent and incubated for 15 min at room temperature (RT). Fluorescence was measured using a PerkinElmer 2103 Envision multilabel plate reader (excitation, 555 nm; emission, 590 nm) and normalized to wells containing cells without S. aureus (0% cell lysis) and cells with 0.05% Triton X-100 (100% cell lysis).

Jurkat A3 cells (2x10⁴/well) were grown overnight in 100 μl of culture medium in 96-well plates. The following day, the cells (measurements were done in triplicate) were pre-incubated for 1 h with the NEDD8 activating enzyme (NEA) inhibitor MLN4924 (MLN; 20 μM; Active Biochemicals Co., Hong Kong, China), the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (zVAD-fmk) (50 μM; Bachem AG, Weil am Rhein, Germany) and the necroptosis inhibitor necrostatin-1 (nec-1; 90 μM; Enzo Life Sciences, Loerrach, Germany). The cells were then incubated overnight with TNFα (100 ng /ml). As necrostatin-1 will cross-react with WST-1, Jurkat A3 cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results were quantified by measuring absorbance at 570 nm using a micro plate reader (TECAN RAINBOW, Germany).