Nunc microwell 96 well flat bottom plate

Sau and Pseudomonas aeruginosa (electronic supplementary material, table S2) were grown for 18 ± 2 h in 5 ml of LB medium at 37°C and 180 r.p.m. Cultures were then diluted in fresh LB medium to contain 2.7 × 10⁶ CFU ml⁻¹. A Nunc Microwell 96-well flat-bottom plate (Thermo Scientific) was seeded in triplicate (from three independent starter cultures) with 75 µl of each of the diluted cultures and 75 µl of 2× working solutions of DHNA, MK-4 or DHNA + MK-4 (prepared from the following stock solutions: 100 mM DHNA dissolved in dimethyl sulfoxide (DMSO), and 50 mM MK-4 (Sigma-Aldrich) dissolved in 100% ethanol). Plate wells contained a final concentration of 10⁶ CFU well⁻¹ with 0, 50 or 100 µM DHNA for growth assays, or with 0 or 100 µM DHNA supplemented with 337 µM MK-4 for growth rescue assays. Plates were incubated at 37°C and 300 r.p.m. for 24 h. Growth was determined by measuring the OD₆₀₀ of the cultures every 30 min during the incubation period. Data from biological replicates were averaged and subtracted from blank data (sterile medium with appropriate additives).

Neutrophils were resuspended in RPMI 1640 (without phenol red) and 10 mM HEPES (Thermo Fisher, Waltham, MA, USA), and seeded (5×10⁴) in quadruplicate in a Nunc™ MicroWell™ 96-well flat bottom plate (Thermo Fisher). Cells were pre-incubated with either 200 nM ruboxistaurin (Selleckchem, Houston, TX, USA), 10 µM diphenyleneiodonium (DPI) (Cayman Chemical, Ann Arbor, MI, USA) or 10 µM dexamethasone (Sigma-Aldrich, St Louis, MO, USA) for 1 h (37°C, 5% CO₂) before stimulation with either LPS (5 µg·mL⁻¹) (Escherichia coli O111:B4) (Sigma-Aldrich), phorbol myristate acetate (PMA) (100 nM) (Sigma-Aldrich) or DMSO control. Neutrophils were incubated for a further 3 h before adding SYTOX™ Green nucleic acid stain (555 nM) (Thermo Fisher) to all wells, to measure extracellular DNA as a surrogate of NET formation. Extracellular DNA was quantified using a fluorescent plate reader (excitation/emission 490/537 nM) and median fluorescence values were reported.