E coli serotype o26 b6

The activity of nitric oxide synthase type II, a master enzyme involved in the inflammatory response, can be monitored in vitro by evaluating the amount nitrite stable metabolites of NO. Nitrite accumulation was measured in the culture medium by Griess reaction. Briefly, The ATDC5 cells were seeded in a 24-well plate at a density of 125,000 cells/well. After overnight starvation, the cells were exposed to a mixture of 25% dialyzed hydrogel and 75% serum-free culture media for 24 and 48 h. Following these periods, 50 μL of the supernatant culture medium was mixed with 50 μL of Griess reagent (equal volumes of 1% sulfanilamide in 5% phosphoric acid and 0.1% naphtylethylenediamine HCl). An absorbance at 550 nm was measured in a microplate reader (MultiscanEX, Thermo Fisher Scientific, USA). Fresh culture medium was used as blank. The amount of nitrite production was calculated from a sodium nitrite standard curve freshly prepared in culture medium. Lipopolysaccharide (LPS) 100 ng mL⁻¹ (E. coli serotype O26:B6, Sigma-Aldrich, USA) was used as the positive control and culture medium from untreated cells as the negative control.

BV2 and MSC were seeded simultaneously at a ratio of 1:0.2 and incubated overnight at 37°C in a 5% CO₂ incubator to allow cells to adhere. Co-cultures were then stimulated with 1 μg/ml lipopolysaccharide (LPS; E. coli serotype O26:B6; Sigma Cat. No. L2762). This culture set-up will be described as ‘activated co-cultures’ hereafter. The time point of LPS addition was considered as 0 hour for all experiments. Cell culture inserts with a 1 μm polyethylene terephthalate membrane pore size (Falcon, BD Biosciences, Erembodegem, Belgium) were used for transwell experiment set-up.

The intratracheal challenge with LPS (E. coli, serotype O26:B6: Sigma Chemical Co.) was performed to simulate bacterial stimuli via an anterior cervical incision to expose the trachea. Animals received the LPS (1 mg/kg diluted in 50 µl of a sterile saline solution) or sterile saline solution (0.9% NaCl; 50 µl)^{32 (link)}. Mice were placed on a heated animal bed (30 °C) until they showed active movements. They were also subjected to post-surgical recovery procedure are received tramadol chlorhydrate (5 mg/mL) via intramuscular injections (Fig. 1). Thus, we did not observe any considerable differences in animal’s behavior. After the recovery period, animals were returned to the cage under normal conditions. The animals were active and did not display signs of post-operative pain.