Stable l glutamine

Neutrophils and macrophages were cultured in 24-well flat-bottom cell culture plates at 5 × 10⁵/well in IMDM medium (Lonza) supplemented with 5% fetal bovine serum (FBS; Lonza), 2 mM stable L-glutamine (Lonza), and 50 mg/mL gentamicin (KRKA, Warsaw Poland) at 37 °C in an atmosphere of 5% CO₂. To determine the influence of selected bacteria and their components on innate immune cell activity, neutrophils and macrophages were stimulated with heat-killed whole bacterial cells (20:1 bacteria per cell), or with their antigens (EPS, LPS, DNA, and PG at selected concentrations, see Figs. 3 and 4 captions). As a reference stimulus, we used 0.1 µg/mL LPS from Escherichia coli strain 0111:B4 (LPS, Sigma-Aldrich). After 24 h of stimulation, culture supernatants were collected and frozen at − 80 °C until use. Cell lysates were used for western blot analysis.

Neutrophils and macrophages were cultured in 24-well flat-bottom cell culture plates at 5 × 10⁵/well in IMDM medium (Lonza) supplemented with 5% fetal bovine serum (FBS; Lonza), 2 mM stable l-glutamine (Cytogen), and 50 mg/ml gentamicin (KRKA) at 37 °C in an atmosphere of 5% CO₂. To determine the influence of the biofilm forms of P. aeruginosa on innate immune cell activity, neutrophils and macrophages were stimulated with heat-killed whole bacterial cells (PAR5-72 h 20:1 and 100:1 bacteria per cell) or with 72-h conditioned media from bacterial cultures (BCM-72 h at 5–20% total volume), if not otherwise stated. The effect was compared with that of bacterial cells (PAR5-8 h) and 8-h conditioned media (BCM-8 h). As a reference stimulus for N1/M1 neutrophils/macrophages, we used 100 ng/ml LPS from Escherichia coli strain 0111:B4 (LPS, Sigma-Aldrich). After 24 h of stimulation, culture supernatants were collected and frozen at − 80 °C until use. Cells were used in a western blot analysis. In some experiments, neutrophils were cultured with BCM-72 h treated with inhibitors of DNA (BCM were incubated in the presence of 2.5 µl of 1500 Kunitz DNAse I, Qiagen; chloroquine, Sigma-Aldrich—2.5 µg/ml) or/and with the inhibitor of LPS (polymyxin B, Sigma-Aldrich, 100 µg/ml) or preincubated with EPS (30 µg/ml) for 1 h and then re-stimulated with LPS (100 ng/ml) or DNA (3 µg/ml).