Escherichia coli lipopolysaccharide e coli lps

At the end of the study peritoneal macrophages were isolated and stimulated ex vivo with 10 ng·mL⁻¹Escherichia coli lipopolysaccharide (E. coli LPS; serotype O55:B5, Sigma Aldrich, St. Louis, MO, USA), which was further purified as described in [30 (link)] or kept in saline as a control for 24 h. Keratinocyte chemoattractant (KC) and IL-6 concentrations were determined in the medium by commercial ELISA kits (Biosource, Camarillo, CA, USA) according to the instructions of the manufacturer.

To address the question whether the obtained conduits activate monocytes, we used the THP1-Blue™ NF-κB reporter cell line, which enables to quantify the induction of NF-κB transcription factor in monocytes. The test was performed according to the procedure described previously [49 (link)]. Briefly, the THP1-Blue™ NF-κB cells were seeded in 24-well plates at a density of 1 × 10⁶ cells/mL; then, the conduits, cut into fragments constituting 1/10 of the well surface, were introduced to each well and incubated for 24 h. Next, 20 μL of supernatants from monocyte cultures were added to 200 μL of Quanti-Blue™ reagent, and the quantification of alkaline phosphatase, as a marker of cell activation (inflammatory response), was measured at 650 nm. The following controls were included: untreated monocyte cultures served as a negative control (cRMPI) and cultures treated with Escherichia coli (E. coli) lipopolysaccharide (LPS) (Sigma–Aldrich, USA) served as a positive control. Furthermore, the commercially available biomaterial Safety-Lok™ blood collection (BCS) served as a reference polymer. Experiments were independently repeated three times, and at least four repeats were included each time.