Sonoplus hd 2070

Tissue samples were ground in small pieces in a mortar with liquid nitrogen and collected in tubes. Tissue lysate was performed incubating the samples for 1 h with a buffer containing 7 M urea, 2 M thiourea, 3 % CHAPS, 40 mM Tris pH 8.3, 1 % ampholytes pH 3–10, protease inhibitors at room temperature. After incubation, tissues were further disrupted with an ultrasonic homogenizer (Sonoplus HD 2070, Bandelin electronic, Germany), and centrifuged at 10,000 x g for 10 min at +4 °C. Supernatant was precipitated by the addition of cold acetone (dilution ratio 1:12 vol/vol) and incubated at −20 °C overnight. After centrifugation at 14,000 x g for 15 min at +4 °C, the pellet was re-suspended and the protein concentration was determined according to the Bradford method. Three pooled samples, both for pathological and healthy gingival tissues, were obtained by mixing equal protein amount of 5 different subjects and were analyzed in duplicate.

The following 4 types of BCG-CWS-loaded liposomal nanoparticles were prepared: Plain liposomes (CWS-L), FA-modified liposomes (CWS-FL), Pep1-modified liposomes (CWS-PL), and FA- and Pep1-modified liposomes (CWS-FPL). Based on an earlier report [6 (link)], a slightly modified LEEL method was employed to encapsulate BCG-CWS into the liposomes. Briefly, BCG-CWS (3 mg) was dissolved in methylene chloride (900 μL), and the organic solution was emulsified with PBS solution (2100 μL) containing liposomal vesicles (6.45 mM phospholipid equivalent) using a sonicator (Sonoplus, HD 2070; Bandelin Electronics, Berlin, Germany). After removing the solvent by rotary evaporation, extrusion was performed with a mini-Extruder (Avanti^® Polar Lipids) using 10 passes through a 200 nm membrane filter. DiI, a red fluorescent probe, was co-encapsulated on purpose for visualizing the cell uptake behavior. Empty liposomes were separately prepared by excluding BCG-CWS or DiI. All liposomal samples were stored in a refrigerator (4 °C) and used in experiments within 3 weeks.

The CNTs were firstly ultra-sonicated in water in a horn sonicator (Sonoplus HD 2070, Bandelin, Berlin, Germany) for 1 h and then added to the cement powder in a weight ratio 2:1 cement–water. Cement paste was reinforced with different content of CNTs according to UNE EN 196-1:2005 [15 ]. A final high speed (18,000 rpm) mixing step was implemented. The fresh cement paste was casted into cylindrical molds of 2 cm height and 1 cm diameter. Plain cement paste was prepared as a control sample. The optimization of the mixing parameters of carbon nanotubes in the cement paste have been selected based on previous studies [16 (link),17 ].

DegraPol polymer solutions of 12 wt% were prepared by dissolving the polymer overnight at room temperature in a mixture of chloroform/HFP (80:20 wt/wt). For emulsion preparation before electrospinning, PDGF-BB was diluted into 0.1% bovine serum albumin (BSA) in MilliQ water at a concentration of 40 μg/mL, while ascorbic acid was dissolved in MilliQ water at a concentration of 50 mg/mL. For water in-oil emulsions, 200 μL of PDGF-BB or AA stock solution were added drop-wise to 5 g of 12 wt% DP polymer solution while stirring at 500 rpm for 2 min. Afterwards, the emulsions were sonicated with a probe ultrasonicator (Sonoplus HD 2070, Bandelin, Germany) for 2 min at 50% amplitude. Immediately afterwards, the emulsion was used for electrospinning.

Solid lipid nanoparticles were prepared by an ultrasonic-assisted hot oil in water emulsion method [45 (link),46 (link)]. The lipid phase composed of Stearic Acid and soy PC was melted under magnetic stirring and kept 10 °C above the Stearic Acid melting point, which is 69.3 °C. A volume of 500 µL of a standard solution of Triamcinolone Acetonide (1 mg/mL in ethanol) was added in the lipid phase. Separately, the aqueous phase containing the non-ionic surfactant (Tween 80) was brought to the same temperature and slowly added to the melted lipid phase. The O/W mixture was kept under magnetic stirring (400 rpm) and at 79 °C for 30 min to create a pre-emulsion and to let the ethanol evaporate, and it was then sonicated in an ice bath for 10 min, with 50% on/off cycles using a probe sonicator (Bandelin Sonoplus HD2070 equipped with a UW 2070 probe, BANDELIN electronic, Berlin, Germany). The resulting solid lipid nanoparticle dispersions were stored at 4 °C until further characterization. All of the tests on the prepared formulation were measured on the same day as the synthesis.

The samples were prepared for analysis using a microwave assisted digestion following the procedure described by Zimmermann et al. [14 (link)]. The weight of fish tissues (muscle, intestine and liver) taken for the digestion ranged between 150 and 340 mg (wet weight). The acanthocephalan weight used ranged between 20 to 130 mg (wet weight) due to expected higher metal levels. The parasite samples were previously pooled for individual fish and subsequently homogenised using a sonifier (Bandelin, Model Sonoplus HD2070). After digestion the clear sample solutions were analysed using inductively coupled plasma mass spectrometry (ICP-MS System Perkin Elmer- Elan 5000) and the concentrations of arsenic (As), cadmium (Cd), colbalt, (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V) and zinc (Zn) were determined (for details regarding instrumentation settings, calibration and sample measurements see Nachev et al. [2 (link)]).
The accuracy of the analytical procedure was verified by analysing dog fish muscle tissue (DORM–3, National Research Council, Canada) as certified reference material. Following analyses, the accuracy rates of seven certified elements were checked.

The method was performed essentially as detailed elsewhere [36 (link)]. Cell monolayers were harvested by scraping into lysis buffer (2% (w/v) SDS, 50 mM Tris-HCl, pH 7.4 supplemented with protease inhibitor [Minicomplete, Roche], 1mM Na₃VO₄, 1mM DTT) followed by 3 × 3 sec sonification (30% amplitude, Sonoplus HD 2070, Bandelin, Berlin, Germany) and centrifugation in a bench-top centrifuge (13.000 rpm, 10 min, 4°C). Supernatants were subjected to colorimetric quantification (Pierce BCA protein assay, Thermo Scientific) and stored at −20°C. SDS-PAGE gels were loaded with equal amounts of protein per lane (25 μg/lane) and transfer was visualized by Ponceau Red staining of nitrocellulose membranes. Primary and secondary peroxidase-coupled Abs (Amersham GE, Little Chalfont, UK) were diluted as recommended by the manufacturers. Chemoluminescence was detected using ECL (Amersham) and quantified in an automated luminescence imaging device (Fusion Solo, Peqlab VWR, Radnor, Pennsylvania).