Cellstar 655180

To determine oxidative enzyme activity, a 50 µL sample was mixed with 150 µL 1 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in citric phosphate buffer, pH 4, in a 96 wells plate (Greiner Bio-One, Cellstar 655180, www.gbo.com). Absorption at 420 nm was measured every min for 30 min with a spectrophotometer (Biotek Synergy HT Reader, Agilent, www.agilent.com). Activity (mM s^-1 L^-1) was calculated using the law of Lambert-Beer with an extinction coefficient of 36,000 M^-1 cm^-1.
H₂O₂ concentration was measured in 5 replicates of SMS and its tea using a hydrogen peroxide assay kit (Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit, A22188, ThermoFisher Scientific Inc, www.thermofisher.com). A spectrophotometer (Biotek Synergy HT Reader, Agilent) was used to measure the fluorescence (Em = 530/25, Ex = 590/35) and concentration was calculated using a calibration curve (0.3–5 µM) based on aqueous H₂O₂ solutions (Sigma-Aldrich). These solutions were also used to determine the stability of H₂O₂ over time.
Viability of microorganisms in SMS tea was tested by inoculating dilution series on LB medium plates. Microorganisms were grown for one week at 25 °C .

CAI4 cells were grown overnight in 5 ml YPD, then diluted to an OD₆₀₀ of 0.1 in YPD containing increasing concentrations of Congo Red (3.125-50 µg/ml) or Calcofluor White (1.5625-25 µg/ml) in a 96-well plate (Greiner Bio-one, CELLSTAR, 655180). The plates were incubated with 400 r.p.m. double orbital shaking in a BMG LABTECH SPECTROstar^Nano plate reader for 48 h at 37 °C in both 0.03 and 5% CO₂. Growth was measured by OD₆₀₀ readings taken every 10 min. The lag times and growth rates for each condition were calculated by fitting the growth curves to a Baranyi model using the DMFit software.

In light experiments for cell aggregation and the migration assay, blue or red light LED panels (225 LEDs, 463 nm 272 μW/cm² or 620 nm 1440 μW/cm²) were used with one or more neutral-density filters (50% reduction). A CLF flora LED module with a controller (CLF Plant Climatics GmbH) was used (463 nm, 20.4 μW/cm²) in the 2D cell clustering assay, different invasion assays, spheroid cultures, the CAM assay and samples prepared for immunofluorescence and western blotting. All the dark samples were continuously wrapped in aluminium foil.
To test the toxicity of the blue light in different experimental set-ups, we seeded 5 × 10⁴ cells/well in 100 μL medium into 96-well microplates (Greiner bio-one, CELLSTAR; # 655 180) and exposed them to different light intensities (0.2–240 µW/cm²) for the maximum duration and under the same conditions as the corresponding experiment. Afterwards, 10 μL of 0.5 mg/mL MTT reagent was added to each well, the cells were incubated for 4 h at 37 °C and 5% CO₂ and then 100 μL of MTT solvent (4 mM HCl and 0.1% NP40 (nonyl phenoxypolyethoxylethanol) in isopropanol) was added to each well. The microplate was then wrapped in aluminium foil and incubated on an orbital shaker for 15 min. Subsequently, the absorbance of the formazan product at 590 nm was measured.