Lta staphylococcus aureus

An amount of 1 × 10⁵ CD14⁺ cells in RPMI were seeded per well into 96-well cell culture plates and incubated for 24 h at 37 °C in either RPMI media alone, RPMI with 1 µg/mL lipopolysaccharide (LPS; Escherichia coli serotype 0111:B4, Sigma, St. Louis, MO, USA), or 10 µg/mL lipoteichoic acid (LTA; Staphylococcus aureus, Sigma, St. Louis, MO, USA). Culture conditions included 5% CO₂. The timing of stimulation was based on prior studies indicating that 24 h stimulation was optimal for monocyte production of innate inflammatory cytokines. After incubation period was over, the plates were centrifuged at 2000 rpm for 10 min after which time supernatant was collected from each well and stored at −80 °C.

CD14⁺ monocytes were seeded in RPMI-1640 (Gibco) with 10% heat-inactivated fetal bovine serum (Omega Scientific; Tarzana, CA), 100 IU/ml penicillin, and 100 IU/ml streptomycin (Sigma, St Louis, MO) at 1.0 × 10⁵ cells/well in 96-well plates. Non-treated (NT) cells served as baseline, treated cells were stimulated with either 1 µg/mL LPS (Escherichia coli serotype 0111:B4, Sigma, St. Louis, MO), or 10 µg/mL LTA (Staphylococcus aureus, Sigma). Cells were cultured at 37 °C with 5% CO₂. After 24 h, cells were centrifuged at 870xg for 10 m and processed for RNA extraction.