96 well black microplate

For fluorescence reading, 250 µl of bacteria and isolated bacterial lysates in Tris buffer (50 mM, pH 7.4) were prepared in 96-well black microplates (Greiner). 2.5 µl of probe 1 (1 mM) was added to test wells, and the same volume of DMSO (1 mM) was added to control wells. The fluorescence intensity (λ_ex = 327 nm, λ_em = 415 nm) was measured at 37 °C in a time-dependent manner by the Victor3 multilabel plate reader (Perkin-Elmer) (0-240 min). For precipitation observations, 1 mM probe 1 with bacteria or bacterial lysates in Tris buffer (50 mM pH 7.4) were prepared in the 96-well white plate (Falcon) and incubated at 37 °C for 24 h.

Kinetic properties were determined by Bio-Layer interferometry (BLI) using Octet RED96 instrument (ForteBio, USA). The operating temperature was maintained at 30°C in 96 well black microplates (Greiner Bio-One, Monroe, USA) which were agitated at 1,000 rpm. For antibody screening, Anti-mouse IgG Capture biosensor tips (AMC) were loaded with each culture supernatants (1:4 dilution) for 5 min in PBS containing 0.1% BSA and 0.02% Tween20. The association of recombinant ΔN-NP protein at concentrations of 300 nM was measured for 3 min, followed by a 5-min-long dissociation phase. All measurements were corrected for baseline drift by subtracting a reference well. Data were analyzed using a 1:1 binding model with local fitting algorithms in the ForteBio data analysis software. For precise affinity measurement using purified monoclonal antibodies, AMC were loaded with 15 μg/mL of each mAbs for 5 min. The association of recombinant Full-length NP protein at concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.781 nM was measured for 3 min, followed by a 5-min-long dissociation phase. All measurements were corrected for baseline drift by subtracting a reference well. Data were analyzed using a 1:1 binding model with global fitting algorithms in the ForteBio data analysis software.

Cytotoxicity assay was performed according to the previously described protocol by Orlando et al. (Orlando et al., 2020 (link)), and it complied with the ISO 10993-5 standard for the test by direct contact. The BC-EAE, as well as empty BC in duplicates, were punched into circular sheets of 5.4 mm in diameter, sterilized by autoclaving at 126 °C for 11 min, and aseptically transferred to HT-29 cells, which had been seeded in a 24-well plate (2 × 10⁴ cells/well and 3 × 10⁴ cells/well), the day before. The cell culture medium (0.5 mL) had been replaced directly before the membrane placement. The cells in a free medium (without BC) were used as the control. After 48 and 72 h incubation in cell culture conditions, the BC-EAE, blank BC, and the medium were removed, and 0.4 mL of fresh medium containing 10% v/v of PrestoBlue™ HS Cell Viability Reagent (Thermo Fisher Scientific, United States of America) was added to each well and then incubated in the same conditions for 30 min. Afterwards, 0.3 mL (3 × 0.1 mL) of the solution from each well was transferred into 96-well black microplates (Greiner, Austria), and fluorescence was measured as described above. The readings were acquired at five independent experiments.