Milli q water

A solution of polymyxin B (sulfate, Sigma-Aldrich, Castle Hill, NSW, Australia; batch number BCBD1065V) was freshly prepared in sterile Milli-Q water (Millipore Australia, North Ryde, NSW, Australia). Enrofloxacin (Sigma-Aldrich) was first dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and subsequently diluted in sterile Milli-Q water to obtain a final DMSO concentration of ≤10% (v/v) (Tran et al., 2016a (link)).

HG 9-91-01 (MedChem Express) was dissolved in DMSO to create a stock solution of 200 mM. Then, with 200-µM aliquots diluted with RNase-free water (Ambion), HG 9-91-01 was injected at concentrations stated in the paper in Figs. 2 and 3. When used with cRNA, HG 9-91-01 was mixed and coinjected. For example, the 200 µM HG 9-91-01 was mixed 1:1 with 2,000 ng/μl cRNA of nonconductive Shaker constructs for injecting 100 µM. DMSO concentration, when injected into the oocyte, was less than 0.1%. Since the volume of an oocyte is ∼1 μl and we injected 50.1 nl, the final concentration of HG 9-91-01 inside the oocyte was ∼4.7 µM.
For synthesis of synthetic melanin with an average diameter of 200 nm, 3 ml ammonia aqueous solution (NH₄OH, 28–30%; Sigma) was mixed with 40 ml of 200-proof ethanol (Decon Laboratories) and 90 ml Milli-Q water (Millipore) with mild stirring at 30°C for 30 min. 0.5 g dopamine hydrochloride (Sigma) was dissolved in 10 ml Milli-Q water and injected into the above solution. The color of the solution immediately turns pale yellow and quickly darkens to brown. The reaction was left at room temperature for 24 h. The melanin mixture was centrifuged and rinsed with copious amounts of Milli-Q water. To create different concentrations, the melanin was dried, and weighed, and resuspended in RNase-free water (Ambion) before injection.

The M09B143 Lacinutrix strain was cultivated in 250 and 400 mL M19 medium in 1 L Erlenmeyer flasks at 10 °C with 140 rpm shaking for 2–3 week until sufficient growth. M19 medium was prepared of 1 L Milli-Q water (Merck Millipore), 20 g D-Mannitol (63560), 20 g Peptone (82303) and 20 g Sea Salt (S9883), all from Sigma-Aldrich. Diaion^® HP-20 resin beads (13607, Supelco Analytica) activated in methanol (34860, Sigma-Aldrich) for 20 min and washed with Milli-Q water were added to the cultures to extract compounds secreted into the medium. After 3–4 days the resin was separated from the cultures by filtrating the cultures under vacuum using a mesh cheesecloth (1057, Dansk Hjemmeproduktion, Ejstrupholm, Danmark). Resin collected on the cheesecloth were washed with 100 mL Milli-Q water and compounds adsorbed to the resin was eluted with methanol. The elution was done twice at 140 rpm for 1 h in 150 mL methanol per 40 g resin. The extract was vacuum filtered through Whatman Ø 90 mm No. 3 filter (Whatman plc), dried under reduced pressure at 40 °C and stored at −20 °C.

Supernatants of BMDM cultures were centrifuged at 410 g for 5 min to remove detached cells and immediately assayed for nitrite ( ${\text{NO}}_2^{-}$ ) as a measure for macrophage NO production. Fifty microliters of supernatant was added to 50 μL of a mixture of 1% sulphanilamide (#S9251, Sigma-Aldrich) and 5% phosphoric acid (#W290017, Sigma-Aldrich) in MilliQ water (Griess reagent A) and incubated in the dark at room temperature for 10 min. Finally, 50 μL of 0.1% N-(1-napthyl) ethylenediamine (#N9125, Sigma-Aldrich) in MilliQ water (Griess reagent B) was added and the absorbance at 540 nm was measured using a microplate reader (BioTek Instruments, Winooski, VT, USA). For each condition, triplicate wells were analyzed and a serial dilution of NaNO₂ (#67398, Sigma-Aldrich) was performed to create a standard curve of ${\text{NO}}_2^{-}$ in the range of 1.56 to 100 μM.

Suspensions of BioPure™ Silver Nanoparticles (AgNPs) of 10, 40 and 100 nm in size, coated with either citrate (CT) or polyvinylpyrrolidone (PVP), were purchased from NanoComposix (San Diego, USA). All the suspensions were supplied at a concentration of about 1.0 mg/ml. BioPure™ AgNPs were chosen because they were guaranteed to be sterile and with an endotoxin level lower or equal to 2.5 EU/ml. The suspending solvents of CT- and PVP-coated AgNPs were 2.0 mM sodium citrate and Milli-Q water (Millipore), respectively. For particle characterization, the CT and PVP-coated AgNPs were diluted with 2.0 mM sodium citrate (cod. W302600, Sigma-Aldrich) buffer and Milli-Q water, respectively. When necessary, samples were sonicated (Elmasonic S 30 H) for up to 30 s, in accordance with the manufacturer’s instructions. In order to prevent contamination, measurements were run using disposable plastic cuvettes. The AgNPs were tested immediately after their delivery and in vivo experiments were run in the following week. In the meanwhile, the AgNPs were stored at +4 °C, according to manufacturer’s instructions.