Transsonic t 460

Dried aerial parts were grounded (10 g), mixed with the 50% (v/v) ethanol in water as solvent, and shaken for 5 minutes for the extraction process. Next, the extraction was performed by ultrasonic-assisted extraction (UAE) using a sonication water bath (Elma Transsonic T 460, Germany) at 35 kHz for 90 min. After these steps, extracts were filtered through the filter paper under vacuum and microfiltered through 0.45 μm pore size microfiltration membrane (Millipore). Nanofiltration was applied to concentrate biologically valuable components and obtain polyphenolic-rich extracts. The nanofiltration was performed with a tangential filtration KOCH Membrane laboratory unit equipped with an NF90 (Dow Filmtec, Sterlitech Company, USA), with a molecular weight cut-off of 200–300 Da, made of polyamide material.

Apple pomace extract as a feed solution was prepared using ultrasonic-assisted extraction (UAE) in a sonication bath (Elma Transsonic T 460, Singen, Germany), at a frequency of 35 kHz for 60 min. The extraction was performed in a 50% (v/v) ethanol solution, and the concentration of the fresh apple pomace was 30% (w/v).

250000 bone marrow derived monocytes were seeded in a 24-well plate Corning OsteoAssay surface plate. Osteoclast differentiation protocol was performed as described above. At the end of differentiation time, cells were washed with 1X PBS for 3 times and incubated with 5% solution of sodium hypochlorite for 10 min at RT to remove the cells. Next, plates were air dried at RT and resorption areas for each well were acquired automatically using Leica DMI6000B. Resorbed areas were quantified with ImageJ and plotted using GraphPad Prism 8.
Alternatively, osteoclasts resorptive capacities were done by a pit assay on bovine cortical bone slices (Immunodiagnostic Systems). 250000 bone marrow-derived cells/well were seeded in a 24-well plate containing the cortical bone slices. At the end of differentiation time, media was removed carefully from the wells by aspiration and bone slices were incubated with ammonium hydroxide solution overnight. Next, cells were removed from bone slices by ultrasonication for 1 min (Transsonic T460, Elma) and slices were stained with 0.5% Toluidine Blue. To visualize resorption pits, bone slices were imaged with Olympus BX41 equipped with Olympus DP72 camera at ×10 magnification. Osteoclast number and total area were quantified with the OsteoMeasure software (Osteometrics, Decatur) and plotted using GraphPad Prism 8.

Neutral lipids were extracted from liver tissues (20–50 mg) according to Folch et al.^{39 (link)}. Briefly, tissues were homogenized in 1 ml of chloroform/methanol (2/1, v/v) using a ball mill (Retsch GmbH). For phase separation, 200 μl of H₂O were added, samples were vortexed for 30 s and centrifuged at 5000 × g for 10 min at 4 °C. Lower organic phase was collected. For repeated extractions, 500 μl of chloroform were added, mixed by vortexing, and centrifuged as above. The lower organic phases were combined and dried in a speed-vac (Labconco). Extracts were dissolved in 0.1% Triton X-100 by incubation for 4 h at 37 °C at 500 rpm in a thermomixer (Eppendorf) and subsequently sonicated in a water bath sonicator (Transsonic T460, Elma). Triglyceride content was analyzed by commercial Infinity Triglycerides Liquid Stable Reagent kit. For protein determination, dried infranatant was dissolved in 0.1% SDS/0.3N NaOH for 4 h and protein content was determined by Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) according to manufacturer’s instructions using BSA as standard.

An in vitro bacterial adhesion assay was performed to quantify the numbers of bacteria that adhered to the extruded tubes. S. aureus was cultured to mid-logarithmic growth phase in TSB at 37 °C and 120 rpm and diluted in TSB to 1 × 10⁶ CFU/mL. The extruded tubes were incubated in 0.5 mL of this suspension in 1.5 mL Eppendorf tubes overnight at 37 °C and 120 rpm. The tubes were gently washed twice with demi water, placed in 1.5 mL Eppendorf tubes with 500 μL of PBS, vortexed 30 s and sonicated at 35 kHz for 15 min in a water bath sonicator (Elma Transsonic T460, Elma, Singen, Germany). This procedure does not affect the viability of the bacteria but releases them from the surface. The sonicates were serially diluted and the number of viable bacteria was determined by quantitative culture on blood agar plates.

The potential antibiofilm synergism of colistin/NAC combinations was investigated using the Nunc-TSP lid system (Thermo Fisher Scientific, Waltham, MA, USA), as described previously [24 (link)]. Briefly, biofilms were grown for 24 h in CAMHB at 35 °C, static conditions. Preformed biofilms were then exposed to three concentrations of colistin (i.e., 8, 32, and 128 µg/mL) and NAC (i.e., 1.6, 8. and 16 mg/mL), alone or in combination. After 24 h of exposure (i.e., 35 °C, static conditions), loosely attached bacteria were removed by two 1-minute washes with 200 µL of phosphate-buffered saline (PBS) (Sigma Aldrich, Milan, Italy). Biofilms were then subjected to 30-minutes sonication (Elma Transsonic T 460, Singen, Germany) in 200 µL of tryptic soy broth (TSB) (Oxoid, Milan, Italy) supplemented with 0.1% Tween 20 (Sigma Aldrich) (i.e., the recovery medium) to remove sessile cells. Mean viable cell counts per peg (CFU/peg) were determined by plating 10 µL of appropriate dilutions of the recovery medium onto tryptic soy agar (TSA) (Oxoid) plates and incubating for 24 h at 35 °C (detection limit, 20 CFU/peg). Colony count was also repeated after 48 h of incubation. Data were obtained in at least two independent experiments, with at least six replicates per condition per experiment.