Spark 20m multimode microplate reader

AHL and HAQ extraction was performed as described previously (127 (link)). Quantification was assessed using Escherichia coli harboring plasmid pSB401 (luxRI′::luxCDABE) and P. aeruginosa PAO1 ΔpqsA CTX-lux::pqsA as biosensors (see Table S2), respectively, by a combined spectrophotometer/luminometer microplate assay. The biosensor strains were grown overnight, and the A₅₈₀ was measured and adjusted to achieve an A₅₈₀ value of 1. For each test well, 5 μL of crude extracts of QS molecules was diluted in 100 μL of LB medium before being added to 100 μL of a 1:50 dilution of the biosensor strains. Further, the bioluminescence and A₅₈₀ were monitored every 15 min for 24 h at 37°C using a Spark 20M multimode microplate reader (Tecan, Männedorf, Switzerland) in white-sided and clear-bottom 96-well microtiter plates. The 3-oxo-C₁₂-HSL, C₄-HSL, HHQ, and PQS synthetic standards (Sigma-Aldrich, Saint-Louis, MO) at final concentrations of 5 μM were added to a 1:100 dilution of the biosensor strains as positive controls. The bioluminescence, recorded as relative light units (RLU), was normalized to the A₅₈₀.

Spark 20M multimode microplate reader (Tecan, Männedorf, Switzerland) was used for measuring fluorescence of sfGFP. The mode Fluorescence Top Reading was used, with the excitation wavelength 485 ± 10 nm and emission wavelength 535 ± 10 nm.

The determination of the antioxidant activity of the extract was performed on the method of FRAP assay [69 (link)], based on the redox reaction of the reduced oxidant Fe(III) complexed by TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine). The FRAP reagent for the calibration curve was prepared mixing 0.3 M acetate buffer (pH 3.6), 0.01 M TPTZ and DI water in proportion 10:1:1, respectively. A 0.001 M FeSO₄·7H₂O solution in DI water was used as standard for the calibration levels. The sample was analyzed mixing 100 µL of the extract solution at 5 mg/mL in DMSO with 900 µL of DI water and 2 mL of FRAP reagent, obtained mixing 0.3 M acetate buffer, 0.01 M TPTZ and a 0.02 M FeCl₃·6H₂O solution in proportion 10:1:1, respectively. All the reagents were prepared fresh. Each solution was prepared in triplicate. All the solutions were incubated for 30 min in the dark. Then the absorbance was measured at 593 nm using a Tecan Spark 20M multimode microplate reader. The scavenging ability was expressed as µmol of Fe²⁺ per 100 mg of extract.

The LDH release assay, a cell death/cytotoxicity assay, plays an important role in assessing the level of plasma membrane disruption. In short, cells with a density of 4 × 10³ were collected and cultured in each well of a 96-well plate for 16 h at 37 °C, under a humidified atmosphere in a CO₂ incubator, and then treated with a series of OCS concentrations (10 μM, 20 μM, 40 μM, or 80 μM) and 0.1% DMSO (as a control) for 24 h. Next, LDH reagent (1:100) was added to each well and incubated in the incubator for 1 h. After incubation, the cells were centrifuge at 400× g for 5 min, and 120 μL of supernatant from each well was transferred to a new 96-well plate, and 60 μL of LDH detection reagent was added to each well. After incubation for 30 min in the dark, absorbance was recorded at 490 nm using a microplate reader (Spark^® 20M Multimode Microplate Reader, Tecan, Switzerland).

For the activity assay, FtAlkBG was overexpressed in E. coli BL21 Star cells (Thermo Fisher Scientific). Cells grew in LB medium supplemented with 100 μM FeCl₃. After growing for 14–19 h at 16 °C, cells were harvested by centrifugation at 5,000g for 10 min at 4 °C. Cells were resuspended in lysis buffer (30 mM Tris-HCl, pH 7.75, 150 mM NaCl, 0.2 mM PMSF, 100 μM ferrous ammonium sulfate hexahydrate and 100 μM sodium hydrosulfite) and lysed using a French press at 13,000 psi. Lysates were centrifuged at 10,000g for 25 min at 4 °C. Pellets were resuspended in lysis buffer without PMSF, frozen in liquid nitrogen and stored at −80 °C until further use.
The dodecane assay was performed on the basis of a modified protocol from McKenna and Coon^{31 (link),48 (link)} using a Spark 20 M multimode microplate reader (Tecan). In a reaction volume of 100 μl, we added 20 μg FtAlkBG, 10 μg PoAlkT and 500 μM NADH in a buffer of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl. The reaction was initiated by addition of 400 μM dodecane dissolved in acetone at 24 °C. The reaction was monitored by measuring the decrease of NADH fluorescence (excitation/emission at 360/460 nm with a bandwidth of 10 nm). Consumption of NADH, that is, decreased NADH fluorescence, is proportional to dodecane hydroxylation to dodecanol. For the control experiment, we added all components except we used acetone instead of dodecane.