Multiskan skyhigh

To evaluate the cell proliferation rate, CCK-8 assay was employed. In brief, AGS and SNU16 cells were added into 96-well plates for incubation. After 24-, 48-, and 72-hour cultivation at 37°C, cells were treated with 10% CCK-8 solution (catalog no. 96992; Sigma-Aldrich, St. Louis, MO, USA) for additional 2 h at 37°C, respectively, and the absorbance at 450 nm was measured using a microplate reader (Multiskan SkyHigh; Thermo Fisher Scientific, Waltham, MA, USA).

MTT assay was used to determine the effect of PSELT or I-PSELT on cell viability following PA exposure. H9c2 cells (5000 per well) were seeded in a 96-well plate and then treated with palmitate (PA) (from 100 to 500 µmol/L) for 24 h or vehicle (BSA) as control. PA was purchased from Sigma and dissolved to make a 10 mM stock solution with 10% fatty acid-free BSA [21 (link)]. Once the concentration for PA-induced cell death was established, H9c2 cardiomyocytes were exposed to PA (100 μmol/L) and PSELT (from 5 to 100 nmol/L) or its inactive form, indicated as inert-PSELT (I-PSELT), from 5 to 100 nmol/L, for 24 h. After treatments, 100 µL of 2 mg/mL MTT solution (Sigma Aldrich) was added to each well after removal of the culture medium, and then cells were incubated for 4 h at 37 °C, 5% CO₂. Finally, MTT solution was replaced by DMSO, and formazan crystals were dissolved. The absorbance was measured at 570 nm using a microplate reader (Multiskan™ SkyHigh, Thermo Fisher Scientific Inc.). The means of the absorbance values of six wells in each experimental group were expressed as the percentage cell viability relative to the control cells. The experiment was repeated three independent times [16 (link),18 (link),19 (link)].

Four hundred mg of each crude polysaccharide (BP, SP and RP) powder was dialyzed (cutoff of 3500), redissolved in deionized water, filtered through a 0.45 μm filter, and applied to a DEAE-Sepharose (Fast Flow, FF) column (50 mm × 40 cm, Beijing Rui Da Heng Hui Science Technology Development Co., Ltd., Beijing, China), as described by Huang et al. [91 (link)]. Briefly, neutral polysaccharide fractions were firstly eluted with deionized water, the elutes were collected, concentrated and lyophilized, and BNP, SNP and RNP were obtained. Then, acidic polysaccharide fractions were obtained after being eluted with a linear NaCl gradient solution (0–1.5 mol/L, 2 mL/min). The elution solution was collected using a computer automatic collector (5 min per tube and 10 mL per tube). Take 200 μL of the elution solution, mix with 200 μL phenol and 1 mL sulfuric acid; cool and transfer 200 μL for determination using UV spectrophotometry (Multiskan SkyHigh, Thermo Fisher, Waltham, MA, USA) with the absorbance value at 490 nm. Then, use absorbance as the vertical axis and the number of tubes as the horizontal axis to create an elution curve, and merge the required parts based on the elution peak. After pooling eluates together based on the elution profile, concentrating and dialyzing (cutoff 3500 Da), acidic polysaccharide fractions were obtained and named BAP, SAP and RAP, respectively.

In vitro biofilms derived from supragingival samples were generated using a modification of the AAA model [26 (link),33 (link)], as previously described [27 (link)]. Briefly, custom stainless-steel lids for 12-well culture plates (VWR) were used to allow the insertion of glass coverslips (18 × 18 mm) (Menzel Gläser, Braunschweig, Germany) [26 (link),27 (link)]. The wells were filled with 3 mL of inocula and, when needed, Aii20J was added to the standardized titer of 20 µg/mL. Sterile Milli-Q water was added to negative control biofilms. After the incubation time, biofilms quantification was performed by CV assay [27 (link)]. After CV staining, biofilms were visually examined [27 (link)]. Absorbance of CV-stained biofilms was measured at 590 nm in a Multiskan SkyHigh (Thermo Scientific, Waltham, MA, USA). Experiments were performed in triplicate.

Adipocyte viability was analyzed by means of XTT assay kit (Sigma-Aldrich, Germany). In 24-well plates, 200 µL adipocytes were mixed with 1.4 mL cell culture medium containing the antibiotics and incubated for 24 h. XTT labeling reagent and electron coupling reagent were added in a 50:1 ratio (800 µL) and samples were incubated for four hours, according to the manufacturers’ recommendations. Subsequently, absorbance was measured at 450 nm using an ELISA reader (Multiskan SkyHigh, ThermoFisher Scientific, Waltham, MA, USA) with a reference wavelength of 650 nm. Absorbance values of the corresponding control group (0.0 µg/mL antibiotics) are considered to be 100%. Results are expressed as absorbance of the test ample in relation to the control and is given as mean ± standard deviation.

Freeze-dried cells were used for carotenoid identification. An aliquot of 100 mg of freeze-dried sample was mixed with 10 ml of methanol, then shaken at 200 rpm at 25°C for 12 h. The resultant methanol extract was centrifuged at 8,000 rpm for 10 min. The UV–Vis absorption spectra were recorded from 200 to 800 nm using a microplate spectrophotometer (Multiskan Skyhigh, Thermo Fisher Scientific). The methanol extract was filtered through a 0.2 μm membrane filter and analyzed using an HPLC system (Shimadzu Corporation, Kyoto, Japan), with a YMC carotenoid (250 × 4.6 mm. D, S-5 μm, YMC) column. The separation column temperature was 35°C, with solvent A consisting of methanol-MTBE-water (85:10:5, v/v/v), and solvent B of 100% MTBE, as the mobile phase. The flow rate was 1 ml/min, the wavelength was set at 450 nm, and a 20 μl sample was injected. For identification of the structure, the sample was five-fold dilution and stored at −20°C until a subsequent analysis by an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) method. HR-ESI-MS spectra were obtained using a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, United States) connected to a quadrupole time of flight mass spectrometer (Xevo G2-XS QTOF, Waters Corp.) from the Korea Basic Science Institute (KBSI, Chuncheon Center).