Victor3 fluorimeter

Chitotriosidase enzymatic activity was determined by a fluorimetric method using 22 μM 4-methylumbelliferyl β-d-N,N′,N″-triacetylchitotriosidase (Sigma-Aldrich S.r.l. Milan, Italy) in citrate-phosphate buffer, pH 5.2, as previously described [21 (link),22 (link)]. Fluorescence was read at 450 nm with a Perkin Elmer Victor3 fluorimeter (excitation wavelength 365 nm, Perkin Elmer, Milan, Italy). As control, cell-free supernatants of monocytes cultured for 24 h were used. Enzymatic activity was measured as nanomoles of substrate hydrolyzed per mL per hour (nmol/mL/h).

HEK293T and HepG2 cells were cultured at 37 °C with 5% CO₂ in Dulbecco’s modified eagle medium containing 10% FBS. Both cell lines were plated individually in 384 well plates (4000 cells per well) and the natural compounds (two fold 10 points serial dilution starting from 5 ppm) were added for 3 days of incubation. After the incubation, 10.0 µL of a 280 µM solution of resazurin sodium salt in water was added to the cells in the assay plate for at least 5 h (final concentration, 40.0 µM of resazurin) and resazurin reduction was measured with a Victor 3™ fluorimeter (PerkinElmer) at 530 nm_Ex/590 nm_Em.

Caco-2 cells were seeded at 5 × 10⁴ cells on the membranes of Thincert cell culture inserts with a 1-μm pore size (Greiner Bio-One) and cultivated for 21 days to achieve differentiation. Overnight-cultured bacteria were harvested, washed once in DPBS, resuspended in growth media for Caco-2 with all the other supplements but no antibiotics, and normalized to an OD₆₀₀ of 0.5. After measuring the TEER value using a Millicell ERS-2 Voltohmmeter (Millipore), 140 μl of LAB [multiplicity of infection (MOI) 800] or 28 μl of ETEC (MOI 40) were added per insert and incubated at +37°C in 5% CO₂. After 24, 48, and 72 h of incubation, the TEER value was recorded. The percentage changes of TEER were calculated by comparing to the baseline (TEER value before adding the bacteria).
When the Caco-2 cells had been incubated with the bacteria as described above for 30 min, 100 μl/insert of FITC-dextran (10 kMW, Thermo Scientific) at the concentration of 300 μg/ml was added to the apical side of the inserts, and the cells were incubated at +37°C in 5% CO₂. After 4, 8, 21, and 48 h of incubation, 50 μl of basolateral medium was collected into a black 96-well plate (Thermo Scientific) and the fluorescence of the FITC-dextran permeated from the apical to the basolateral side was measured using a Victor3 fluorimeter (Perkin Elmer).