Thp1 dual monocytes

THP1‐Dual monocytes (Invivogen) were maintained in RPMI 1640 (ThermoFisher Scientific) medium with 10% Fetal Calf Serum (FCS), 25 mM HEPES, and 2 mM l‐glutamine at 37°C, 5% CO₂. For experiments with bacteria, THP1‐Dual cells were seeded in a 96‐well plate at a concentration of 10⁵ cells/well. Bacteria were added to the cells at 10⁶ CFU/well for live bacteria from cultures, 10⁷ CFU/well for UV‐inactivated bacteria from cultures, and 10⁸ CFU/well for powdered bacteria. Spray was added at a 1:20 dilution. The plate was incubated for 24 hours at 37°C and 5% CO₂. Induction of NF‐κB was assessed based on SEAP reporter activity at 405 nm with the Synergy HTX Plate Reader (BioTek) after the addition of a para‐nitrophenylphosphate (pNPP) buffer. Induction of IRF was assessed based on luciferase reporter luminescence activity with the Synergy HTX Plate Reader (BioTek) after the addition of the QUANTI‐Luc™ (InvivoGen) buffer. Poly (I:C) with Lipofectamine 2000 (Invitrogen) at 50 μg/ml for IRF induction or lipopolysaccharides (LPS) from E. coli (Sigma) at 20 ng/ml for NF‐κB induction were used as positive controls.