Synergy neo2

The total flavonoid content (TFC) was determined according to the aluminum chloride colorimetric method with some modifications [15 (link)]. Briefly, OL extracts and standards (catechin) were mixed with sodium nitrite for 6 min in a 96-well plate. Then, aluminum chloride was added and incubated at room temperature for 5 min in the dark. Finally, sodium hydroxide was added to each well, and the absorbance was measured at 510 nm in a microplate reader, Synergy Neo2 (Winooski, VT, USA). Results were expressed as mg of catechin equivalents (CAT)/g of DW extract. Data are mentioned in tables as “mean ± SD”.

Each sample was prepared in triplicate to a final volume of 10 μl in 50 mM HEPES, pH 7.5, 10 μM nickel chloride with 10 μM enzyme and 10 mM PNAG hexamer and incubated for 24 hr at 20°C. To correct for the presence of free amines on the enzyme, each tested enzyme was added to control samples at a final concentration of 10 μM just prior to reaction with fluorescamine. The reaction was performed by adding to each 10 μl sample, 20 μl of 0.5 M borate buffer, pH 9.0, and 10 μl of a freshly prepared 20 mg/ml fluorescamine solution in dimethylformamide and mixed by pipetting. The reaction was then allowed to stand at room temperature for 10 min before adding 80 μl of deionized water. An 80 μl aliquot of the solution was removed from each sample and transferred to a Corning 3686 half-area 96-well plate for measurement in a Synergy Neo2 plate reader (360-nm excitation, 460-nm emission, 5-nm slit widths). Glucosamine solutions were used as standards to calculate amine concentration.

The measurement of integrity of the epithelial tight junction was performed by the FITC-dextran flux assay, as previously described [28 (link)]. T84 cells were seeded on a Transwell permeable support and cultured for 7 days to develop monolayers. T84 cell monolayers were treated with the vehicle, afatinib (10 µM), or COS (100 µg/mL) either alone or in combination for 24 h. Following treatment, FITC-dextran was added into the apical media (1 mg/mL) and, one and a half hours later, basolateral media were sampled for the determination of FITC-dextran concentrations using a Synergy/neo2 multi-mode reader.

P. aeruginosa strains were grown at 37°C shaking in LB overnight prior to growth curve experiments. The following morning, cultures were back-diluted to an optical density at 600 nm (OD₆₀₀) of 0.01 in ASMi in 10-ml glass tubes with 2 ml medium (for fluorescence growth curves) or 50-ml Falcon tubes with 30 ml of medium (optical density growth curves), rotating at 250 rpm on an incubated orbital shaker at 37°C. C. albicans strains were grown at 30°C shaking in YPD overnight prior to growth curve experiments. They were cultivated overnight at 30°C to maintain cells in yeast form prior to the start of growth curve or biofilm experiments (see below). The following morning, cultures were back-diluted to an OD₆₀₀ of 0.01 in ASMi in 10-ml glass tubes with 2 ml medium (for fluorescence growth curves) or 50-ml Falcon tubes with 30 ml of medium (optical density growth curves), rotating at 250 rpm on an incubated orbital shaker at 37°C. Fluorescence measurements were made using a Synergy Neo2 every 6 h. A 543-nm excitation source was used to excite mKO-κ, and a 594-nm excitation source was used to excite mKate2. Optical density measurements were made every hour using a benchtop spectrophotometer (CWA Biowave CO8000 cell density meter).

We assessed ADA2 enzyme activity in patients’ serum and cell cultures using a commercial kit for ADA2 enzyme activity (Diazyme Laboratories). Peripheral blood was collected and serum was separated by centrifugation. Enzyme activity was detected by plate reader (Synergy NEO2). ADA2 activity was isolated from total ADA activity by inhibiting ADA1 with erytro-9-(2-hydroxy-3-nonyl) adenine (EHNA, 1 mg/mL).

Cells were incubated with 5 μM DHE (in RPMI) for 15 min in the dark at 37 °C. Cells were then washed once with cell-based assay buffer, and red fluorescence was recorded by Synergy Neo2 multi-plate reader.