Synergy htx plate reader

Single colonies from LB plates were inoculated in 400 μl of EZ-RDM (Teknova) supplemented with the appropriate antibiotics and grown in 96-deep-well plates at 37°C with shaking overnight 900 rpm on a Heidolph titramax 1000. 100 μl of the overnight culture were transferred into flat, clear-bottomed black 96-well plates (Corning) and the OD₆₀₀ and fluorescence were measured in a Biotek Synergy HTX plate reader. For mRFP1 (referred to as mRFP) detection, the excitation wavelength was 540 nm and emission wavelength was 600 nm. For sfGFP detection, the excitation wavelength was 485 nm and emission wavelength was 528 nm. For mTagBFP2 (referred to as mBFP) detection, the excitation wavelength was 400 nm and emission wavelength was 450 nm. At least three biological replicates were used for all experiments unless stated otherwise.
For kinetic experiments, overnight cultures in stationary phase were subcultured to OD₆₀₀ 0.1 in 200 μl of EZ-RDM (Teknova) supplemented with the appropriate antibiotics. Cultures were grown in flat, clear-bottomed black 96-well plates (Corning) at 37°C in a Biotek Synergy HTX plate reader set to shake at 1200 RPM. Surrounding wells were filled with 200 μl of water to maintain humidity. The OD₆₀₀ and fluorescence were measured every 30 min for 16 h.

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.