Spectramax i3x plate

PrecA-LUX^{33 (link)} was grown to an OD₆₀₀ of ∼0.4,
diluted 10-fold in 7H9/OADC/Tw, and inoculated into white, clear-bottom,
96-well microtiter plates (Greiner CellStar) containing two-fold serial
dilutions of the drug. The plates were incubated at 37 °C, and
luminescence was recorded every 24 h for 8 days using a SpectraMax
i3x plate reader (Molecular Devices). Data were plotted in Prism 9
(GraphPad).

Solutions of 50 μM 9 (diluted from the stock) and 0–150 μM and ZnCl₂ (diluted from a 5 mM stock solution in 10 mM Tris buffer, pH 7.5) were prepared in 10 mM Tris buffer, pH 7.5 to a final volume of 500 μL. Each solution was mixed well, then 150 μL was distributed to each of three wells in a transparent flat bottom, low volume, acrylic 96-well plate. The UV-Visible spectrum was recorded for each well of the plate by a Molecular Devices SpectraMax i3x plate reader. Spectra were collected from 230 nm to 900 nm every 1 nm. Spectra were averaged for the three wells containing identical solutions.

Bacterial cells were grown to the exponential phase (approximately OD₆₀₀ = 0.6) and resuspended in reaction buffer (20 mM Tris-Cl, pH 8.0). The purified endolysin (LysGR1) and its EAD were added to a final concentration of 1.6 μM, and the OD₆₀₀ values were monitored over time at 45°C using a SpectraMax i3x plate reader (Molecular Devices, Sunnyvale, CA). The relative lytic activity was calculated when the OD₆₀₀ of the endolysin-treated group (experimental OD₆₀₀) reached the lowest value using the equation as follows: $\frac{ControlO{D}_{600}-ExperimentalO{D}_{600}}{InitialO{D}_{600}}\times 100\left(\%\right)$
The effect of pH on the enzymatic activity of LysGR1 and LysGR1_EAD was assessed, as previously described (Ha et al., 2018 (link)), yet with some modifications. An amount of 1.6 μM of endolysin was added to Bacillus amyloliquefaciens KACC 15877 cells suspended in Britton–Robinson universal buffer (0.04 M H₃PO₄, 0.04 M H₃BO₃, 0.04 M CH₃COOH, and 0.2 M NaCl) with various pH buffers, which ranged between 6 and 10. To evaluate the effect of the temperature on LysGR1 and its EAD lytic activity, preincubated LysGR1 and its EAD at different temperatures (−20–80°C for 15 min) were used. The effect of NaCl on the enzymatic activity of LysGR1 and its EAD was tested with the reaction buffer using various NaCl concentrations (0–1,000 mM).

RAW264.7 cells were cultured for 4 h in black 96-well plates at a density of 1 × 10⁵ cells/well and then treated with β-glucan or Euglena for 48 h. NO production was measured using a Nitric Oxide Synthase Kit obtained from Sigma‒Aldrich (USA) according to the manufacturer’s protocol. The fluorescence intensity was measured using a SpectraMax i3x plate reader (Molecular Devices, USA) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

After meconium digestion, we allowed meconium solids to sediment on the benchtop for 30 min. We measured the release of colored pigments from the meconium by sampling 200 µl of the supernatant and transferring it to a clear 96 well plate (Corning). We obtained the absorbance spectrum of the supernatant for each sample using a SpectraMax i3x plate reader (Molecular Devices) recording wavelengths between 300 and 700 nm at 5 nm intervals. We used the corresponding test solutions as blanks for each sample. Because the peak in meconium pigment absorbance was near 405 nm, we used A₄₀₅ to compare pigment release between different conditions. We used 10 pigs to compare test solutions (5 males, 5 females, all CFTR^−/−).