Sodium nitrite

NO production was monitored by measuring nitrite content in the culture medium as previously described [32 (link)]. Samples were mixed with Griess reagent (Sigma, St. Louis, MO, USA) and a standard curve was constructed using sodium nitrite (Sigma, St. Louis, MO). The absorbance at 548 nm was measured using an ELISA microplate reader (Spectramax, Molecular Devices) after incubation for 30 min.

EBC samples collected for a previous study^{53 (link)} were utilized. The details of the sample collection procedure have been previously described, but briefly, 1–2 mL of EBC was collected during 20 min of tidal breathing from each of seven adult subjects using an EcoScreen device (Jaeger, Wurzburg, Germany), which condensed the exhaled breath at −20 °C. All surfaces were triple-rinsed with nitrite-free water prior to contacting the EBC, and the samples were frozen at −80 °C for later analysis. In the previous study, nitrite concentrations were measured using selective catalytic reduction and chemiluminescence detection (NOA 280i, GE Analytics, Boulder, CO, USA)^{53 (link)}. All solutions were prepared with distilled water. For testing and calibration of the sensors, we experimented with various buffers, including sodium nitrite, acetate, and phosphate buffers (Sigma-Aldrich, St Louis, MO, USA).

The concentration of NO in the culture medium was determined using the Griess reagent. A volume of 80 μL of the culture media collected after the exposure was pipetted into 96-well plates. A stoichiometric solution (v/v) of 1% sulphanilamide (Sigma-Aldrich, St. Louis, MO, USA) and 0.1% dihydrochloride N-(1-naphthyl) ethylenediamine (Sigma-Aldrich) was prepared just before and it was added to wells in a ratio of 1:1 (v/v). The absorbance of the colored product formed was measured spectrophotometrically at 550 nm at the Flex Station plate reader (Molecular Devices, San Jose, CA, USA), and the NO concentration in the samples was determined by extrapolation on a standard sodium nitrite (Sigma-Aldrich) curve with concentrations between 3125 and 100 μM, which was carried out under the same conditions.

Sucralfate, lipopolysaccharide (LPS), ferric chloride, trichloracetic acid (TCA), sodium nitrite, potassium ferricyanide, dimethyl sulfoxide (DMSO), N-(1-naphthyl) ethylenediamine dihydrochloride, phosphoric acid, and sulfanilamide were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), the antibiotic mixture (penicillin-streptomycin), and fetal bovine serum (FBS) were purchased from Hyclone (South Logan, UT, USA). All chemicals not listed were grade of reagent.

Bc407 was grown in LB medium at 37 °C with agitation (200 rpm) until the end of the exponential growth phase (OD₆₀₀ = 2). Then, bacteria were diluted 1/100 in RPMI 1640 Medium GlutaMAX^TM (SKU 61870-010, Thermofisher), and exposed to diverse stresses for 15 min to 60 min at 37 °C without agitation.
NOC5 (146724-82-5, Calbiochem) was used as NO donor at a final concentration of 10 and 50 µM. Sodium nitrite (7632-00-0 Sigma-Aldrich) was used at 2.5 mM, Potassium nitrate (7757-79-1, Merck) at 20 mM and hydrogen peroxide solution (7722-84-1, Sigma-Aldrich) at 0.03% (V/V). The quantity of nitrate in solution was measured using the Griess Reagent System (G2930, Promega) following the manufacturer protocol.
Bacteria were starved from iron during incubation in iron-free RMPI medium for one hour at 37 °C with agitation. Then iron stresses (excess or starvation) were induced during 15 min with Iron III citrate (6100-05-6, Sigma-Aldrich) at 81 µM, and 2,2′-Bipyridyl (366-18-7, Sigma-Aldrich) at 4,4 μM as an iron chelator, respectively [25 (link),26 (link)].

All chemicals used in the analysis, such as sodium tripolyphosphate, sodium erythorbate, sodium nitrite, bathophenanthroline, potassium iodide, and cholroform, were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA).