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Growth curves were conducted in 50% MRS. Overnight cultures of isolates and the wildtype were diluted 500-fold in 50% MRS medium. For each culture, 300 µL was transferred to a 96-well cell culture plate (Sterile, F-bottom, with lid, Greiner bio-one, Cellstar) and grown under static conditions at 30 °C in a BioTek ELx808 (BioTek) using the Gen5 software (version 2.05) with OD₆₀₀ measurements every 20 min. Growth curve data was analyzed based on the kinetic measurements using a modified version of the GrowthCurver package^{23 (link)}, allowing for the occurrence of blank observations in the dataset. For culture yield data, samples were homogenized by pipetting post incubation and 250 µl was transferred to a new 96-well plate before measurement of optical density OD₆₀₀. Four biological replicates were conducted to assess culture yield and growth rate (generation time).

HPMECs (1.5 × 10⁵ cells/well) were seeded on the 24-well Transwell system (Greiner Bio-One, 0.4-mm pore size, 6.5-mm diameter, transparent, Costar,The Netherlands). The cells were cultured for 4 days and the media was replaced with serum-starved medium for 2 h before exposure to any inhibitor/stimulator. The endothelial cell barrier function was evaluated by measurements of transendothelial electrical resistance (TEER) across confluent cells utilizing Millicell-ERS (MERS00002, Millipore, Bedford,USA). Transendothelial permeability was analyzed using Chemicon’s in vitro vascular permeability assay, which utilizes fluorescein isothiocyanate–dextran (FITC-dextran, 40-kDa, Sigma, USA) [20 (link)]. The Transwell plate was removed from the incubator to quantify the passage of FITC-dextran across the monolayer. Using a micropipetter, media (50 μl) was removed from the bottom chamber of each Transwell and transferred to a 96-well plate. The 96-well fluorescence plate reader was used at an excitation wavelength of 488 nm and emission wavelength of 515 nm. Data was represented graphically as raw data in the arbitrary fluorescence units generated by the plate reader.

Wells of microtiter plates (Greiner Bio-One, Sigma-Aldrich) were coated with 200 μL anti-rCsGSTo1 or 2 antibodies (1.0 μg/mL) in a carbonate–bicarbonate buffer (100 mM, pH 9.6) overnight at 4 °C. The plates were blocked with phosphate buffered saline containing 0.05% Tween 20 (PBS/T) and 1% bovine serum albumin (BSA) for 1 h at 37 °C, after which, 200 μL rat biliary epithelial extracts (10 μg/mL) were incubated for 2 h at 37 °C. The plates were incubated with 1:1000 diluted C. sinensis-infected rat sera (200 μL) for 1 h and subsequently with horse radish peroxidase-conjugated goat anti-rat IgG (1:2000 dilution) for 1 h at 37 °C. Color reaction was developed with Ultra TMB-ELISA substrate (200 μL/well; Thermo Fisher Scientific) in the dark for 10 min and stopped by adding 4 N H₂SO₄ (50 μL/well). Absorbance was read at 450 nm. PBS/T was used as a blank control. All results were measured after an appropriate blank correction. Each sample was assayed in triplicate.

The quantitative turbidimetric assay was used for the measurement of IgG in test samples [25 ]. The anti-rabbit IgG antibodies formed insoluble complexes when mixed with samples containing purified rabbit IgG (Invitrogen, Life technologies, Carlsbad, CA). The scattering of light by the immune-complexes to determine the IgG concentration in the sample was quantified by comparing with a calibrator of known IgG concentration (5 mg/ml to 0.625 mg/ml). The 50μl volume of each dilution was dispensed in duplicate in 96 well flat bottom microtitre plate (Greiner bio-one, Cellstar) and 50μl of anti-rabbit IgG prepared in tris buffer was added to these dilutions. Absorbance of each well was measured at 540 nm by ELISA plate reader (Biotek instruments, powerwave X2, USA) and plotted between absorbance and IgG concentration of each dilution. The IgG standard curve was used to determine the concentration of IgG in test samples.

10⁵ cells were added per well in 96-wells round bottom plates (Greiner bio-one, Cellstar) and stained with 10 µg/ml mouse anti-CD89 mAbs or a mouse IgG₁ negative isotype control (Biolegend). Subsequently, PE-labeled goat-anti-mouse IgG antibody (Jackson) was added. After final washings, cells were fixed in PBS/0.1% bovine serum albumin (BSA; Fitzgerald)/2%formaldehyde (37%; Sigma). Binding of anti-CD89 antibodies was determined with flow cytometry (FACS Cyan, Beckman Coulter), and expressed as signal-to-noise (S/N) ratios, ie, dividing measured geometric fluorescence intensity of anti-CD89 antibody by measured geometric fluorescence intensity of mouse IgG1 isotype control.

Isolated PMNs were resuspended in 10%RPMI++. 2x10⁵ cells were seeded per well in 96-wells round bottom plates (Greiner bio-one, Cellstar) and incubated with 20 µg/ml anti-CD89 mAbs or a mouse IgG₁ isotype antibody as negative control (Biolegend) for 20 minutes (min) at 4°C. Green fluorescent latex beads coated with BSA or IgA were incubated with the cells for 30 min at 37°C a cell-to-bead ratio of 1:60. After washing, fluorescence intensity was measured with flow cytometry (BD FACS Calibur, BD bioscience) and phagocytic index was calculated as the percentage of cells that phagocytized, multiplied by the mean fluorescent intensity of bead-positive cells (6 (link)).

B. subtilis strains were grown overnight in 100 µl LB medium supplemented with appropriate concentrations of antibiotics in 96-well plates at 37 °C under vigorous shaking at 1000 rpm in a Thermo Shaker L079-100 (Kisker). An over-day culture in 96-well plates was inoculated with the over-night culture at a 1/20 dilution and grown under the same conditions for 6 h. Cultivation of B. subtilis was carried out in a 96-well plate with flat bottom (Greiner bio-one, Cellstar) in a final volume of 100 µl of LB or EnPressoB medium (BioSilta), inoculated with 5 µl from the over-day culture. To avoid evaporation all plates were covered with a lid, incubated at 37 °C with constant shaking (fast mode: 1140 rpm) using a Biotek Synergy two multimode microplate reader. Fluorescence (excitation 485/20 mm, emission 528/20 mm) and optical density (OD₆₀₀) were measured at 10 min intervals. For light path length correction, the OD₉₇₇ and OD₉₀₀ were measured and were calculated for a sample length of 1 cm by (OD₉₇₇–OD₉₀₀)/0.18 as described previously [34 (link)].
GFP-expression levels were corrected for background fluorescence by subtracting the fluorescence values derived from the cultivation of the wild-type strain 168 (cultivated in six independent wells). Next, normalization of the fluorescence increase per time and growth rate was calculated using the following equation: (dGFP/dt/OD₆₀₀).