Lps from escherichia coli 0111 b4 l4391

Bortezomib (sc-217786), azithromycin (sc-254949), minocycline (sc-203339) and SB-3CT (sc-205847) were purchased from Santa Cruz Biotechnology (TX, USA). LPS from Escherichia coli 0111:B4 (L4391) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human full-length proMMP-9 (92 kDa) was expressed in Sf9 insect cells and purified by gelatin-Sepharose chromatography. ProMMP-9 was activated by incubation with the catalytic domain of stromelysin-1/MMP-3 (cat. No. 444217, Merck Millipore, Darmstadt, Germany). The recombinant expression, purification and activation of proMMP-9 were performed as described previously [14 (link), 35 (link)].

Peripheral blood mononuclear cells were purified from the blood of healthy donors by density gradient centrifugation on Ficoll-sodium diatrizoate (Lymphoprep, Axis-Shield PoC AS, Oslo, Norway). Next, CD14⁺ monocytes were isolated using the EasySep™ human CD14 positive selection kit (StemCell, Grenoble, France). Cells were seeded in 24-well plates at a density of 2 × 10⁶ cells/ml and SLPI or SLPI digests were added at a concentration of 1 µg/ml. After 30 min incubation at 37°C, cells were stimulated with 1 µg/ml LPS from Escherichia coli 0111: B4 (L4391, Sigma-Aldrich, St. Louis, MO, USA). After 24 h, cells and supernatants were collected.