Powerwave 340

The quantity of 50 µL of plasma was analysed in duplicate. Detection of LPS was performed using an ELISA Kit for Lipopolysaccharide (LPS) (Cloud-Clone Corp, CCC, Katy, TX, USA) according to the manufacturer’s recommendations. Absorbance analysis was carried out using a PowerWave 340 (BioTek) and Gen5™ software.

The strains used in the current study were obtained from the Japan Collection of Microorganisms (JCM; Ibaraki, Japan) and Yakult Culture Collection (YIT; Tokyo, Japan). The strains were routinely cultured at 37°C in an anaerobic chamber (Coy Laboratory, Grass Lake, MI, USA) with 88% N₂, 5% CO₂, and 7% H₂, using mGAM broth (Nissui Pharma, cat. 05422) containing 0.5 w/vol% glucose and 0.5 w/vol% lactose. Evaluation of Bifidobacterium carbohydrate utilization profiles was performed at 37°C in modified ILS-PIPES (100 mM PIPES, pH 7.1, 5 g/L yeast extract, 10 g/L trypticase peptone, 3 g/L tryptose, 1 mL/L Tween 80, 0.3 g/L l-cysteine hydrochloride, 575 mg/L MgSO₄･7 H₂O, 154.5 mg/L MnSO₄･5 H₂O, 34 mg/L FeSO₄･7 H₂O, and 2 g/L diammonium hydrogen citrate) supplemented with the targeted carbohydrates (0.5 w/vol%). Growth curves were evaluated by measuring the OD₆₀₀ every 30 min using a microplate reader PowerWave 340 (BioTek, Winooski, VT, USA) in an anaerobic chamber.

Glucose samples other than those analyzed via glucometer, as well as triglycerides and total cholesterol, were analyzed using a Vet Axcel clinical chemistry system (Alfa Wasserman, Diagnostic Technologies LLC, West Caldwell, NJ, USA). Fasting insulin was analyzed using a Mouse Ultrasensitive Insulin ELISA kit (Alpco, Salem, NH, USA) using a PowerWave 340 microplate reader (BioTek, Winooski, VT, USA). Samples were analyzed in duplicate.

ELISA was performed for NF-κB p65 ELISA Kit (Cat. No. ab176663, Abcam, Burlingame, CA, USA), following the manufacturer’s protocol. Briefly, 500 μL of cell culture supernatants were centrifuged at 2000× g for 5 min to remove particulates before being collected for further processing. Measurement of soluble factors in cell culture media was completed on collected supernatants from three independent experiments. The optical density was measured at 450 nm using a BioTek PowerWave 340 microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Concentrations of the cytokine in the samples were determined using a standard curve of known concentrations from the standard sample provided by the kit. For each independent experiment, at least two technical replicates were combined for analysis.

Six disk-shaped samples (1 mm thickness and 7 mm diameter) from each material (n = 6) were prepared using silicone molds, and individually light-cured for 40 s. The samples were stored in 2 mL distilled water, and storage solutions were exchanged after 28 and 45 days with equal volume replacement. The calcium release was determined by mixing the storage solutions with Arsenazo III in 20 mM HEPES at pH 7.4 (Sigma Aldrich, St. Louis, USA). The analysis of calcium release through this solution was performed using a UV-Visible spectrophotometer (Powerwave 340; Biotek, St. Paul, USA) with 656 nm wavelength for 3 s adopting the Arsenazo III colorimetric method [21 (link)]. Aliquots of 5 μL of the samples (diluted 1 : 10 and partially neutralized) were added to 50 μL of deionized water before UV-Vis analysis. For calibration, standards containing 40 to 200 μg Ca/mL solutions (Sigma Aldrich) were used.

The BBB disruption (BBBD) can be quantified based on the extravasation of EB, which binds to albumin. This has been used previously for the evaluation of vascular permeability induced by focused ultrasound [27 (link)]. In this study, we used EB to assess the relationship between behavioral alterations and BBB integrity after FUS sonication. The rats were injected intravenously with EB (Sigma, St. Louis, MO) at a concentration of 100 mg/kg at 0 and 24 hours after FUS application. The animals were sacrificed approximately 4 hours after the EB injection. Animals were perfused with saline via the left ventricle until colorless perfusion fluid appeared from the right atrium. After perfusion and brain removal, the brain was sectioned into three slices from 0 to 6 mm posterior to the bregma and these were mounted on glass slides. The coronal sections were then divided into right and left hemispheres before the amount of EB extravasated was measured. Samples were weighed and then soaked in 50% trichloroacetic acid solution. After homogenization and centrifugation, the extracted dye was diluted with ethanol (1:3), and the amount of dye was measured using a spectrophotometer (PowerWave 340, BioTek, USA) at 620 nm. The content of EB in the tissue was quantified using a linear regression standard curve derived from seven concentrations of the dye, and was denoted per gram of tissue.

Cell viability was measured by quantitative colorimetric MTT assay [35 (link)]. Briefly, SH-SY5Y cells (1 × 10⁵/mL in a 96-well plate) were plated with culture medium and incubated for 24 h at 37 °C, with 5% CO₂ in a humidified atmosphere. The cells were then incubated with test compounds at various concentrations for various times. The reaction was stopped by removing the treatment media, adding MTT reagent (0.1 mg/mL), and then allowing the reagent to react at 37 °C in 5% CO₂ for 2 h. MTT was removed, and cells were lysed with DMSO. The absorbance at 570 nm was measured using an ELISA reader (PowerWave 340, Bio-Tek Instruments, Winooski, VT, USA). The cell viability (%) was calculated using the following Equation: $\mathrm{Cell}\mathrm{viability}(\%)=(\frac{\mathrm{T}}{\mathrm{C}})\times 100$
where T is the absorbance in the test, and C is the absorbance for the control.