Sonopuls hd 2070

Polyclonal rabbit antibodies were produced by Davids Biotechnology (Regensburg, Germany) using recombinantly expressed and purified TFB-RF1 (Ochs et al., 2012 (link)). The IgG fraction of the polyclonal antibodies was purified using an immobilized Protein G column (GE Healthcare, Little Chalfont, United Kingdom). Antibody containing fractions were pooled and dialyzed overnight against phosphate-buffered saline (PBS). For the preparation of cell extracts from the Pyrococcus strains, cell cultures with a volume of 20 ml and a cell density of approximately 1 × 10⁸ cells per ml were harvested, washed with 1 ml PBS and resupended in 300 μl PBS including protease inhibitor mix (cOmplete Ultra Tablets, Roche Applied Science, Mannheim, Germany). Cells lysis was induced by sonication (Sonopuls HD2070, Bandelin Electronics, Berlin, Germany), cell debris was removed by centrifugation and the protein concentrations of the supernatants were determined by Bradford assay. Western blot experiments were done as previously described (Waege et al., 2010 (link)). The signals were visualized using a Cy5-labeled secondary anti-rabbit antibody from Thermo Scientific (Waltham, MA, United States) and a fluorescence image analyzer (FLA-5000, Fuji, Japan). Each experiment was repeated three times and representative data are shown in the manuscript.

The high-energy ultrasonication technique was used to prepare nanoemulsions containing propolis oily extract. First of all, propolis oily extract was homogenized with surfactants (such as Tweens and Spans). The dose of propolis was adjusted according to the amount of polyphenol in it, and it was in the form of 25 mg of polyphenol in 758.33 mg of propolis oily extract. Then, 250 mg of dexpanthenol was added to the water phase. The water phase was added to the oil phase, and ultrasonication (Bandelin Sonopuls HD 2070, Berlin, Germany) was applied for a certain time (100% amplitude, cycle 3), and nanoemulsions were formed (n = 6) [59 (link)]. Blank nanoemulsions were prepared according to the method described above without adding propolis and dexpanthenol.

Freeze-dried filters containing cyanobacterial material and the cell-bound fraction of MCs were extracted with 2.4 mL of 75% methanol for MCs (Spoof et al. 2003 (link), Hautala et al. 2013 (link)). The extracts were sonicated for 15 min in a bath sonicator (Bandelin Sonorex RK 156, Berlin, Germany) and additionally for 1 min with a probe sonicator (Bandelin Sonopuls HD 2070 with a 3-mm microtip, 30% pulse, 30% energy). After centrifugation at 10,000g for 10 min, part of the supernatant was concentrated by evaporation with nitrogen gas at 50 °C. The residue was resuspended in 75% methanol (150 μL) and clarified by filtration through an Acrodisc GHP 0.45 μm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA) prior to MC analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
The OASIS HLB SPE cartridges prepared at the site of sample collection and intended to capture to the extracellular MCs were eluted with 3 mL of 100% methanol. The methanol was evaporated with nitrogen gas at 50 °C. The dry residue was resuspended in 75% methanol (150 μL) and clarified by filtration through an Acrodisc GHP 0.45-μm syringe filter prior to LC-MS/MS.

The P81 gene product was synthesized and cloned into pET21a(+) by Biomatik (https://www.biomatik.com/). For protein production, inoculated broth cultures (50 ml in a 250 ml flask) were cultured at 37 °C until OD_600nm of 0.5–0.6 was reached. Protein expression was induced through addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and cultured overnight at 37 °C with shaking (150 rpm). Cells were harvested by centrifugation at 4 °C, 3265×g for 5 min. The pellet was resuspended in 20 mM Tris–HCl (pH 7.9), 50 mM NaCl and sonicated on ice using a Bandelin Sonopuls HD 2070 (58% power, 5 cycles for 30 s). The sonicated cell debris was collected by centrifugation at 4 °C, 13 000×g for 20 min. The soluble fraction was filtered through a 0.45 µm syringe filter and purified using nickel affinity chromatography. The purity of the protein was analysed on a 12% SDS-PAGE stained with Coomassie (Additional file 1: Figure S1). The protein was concentrated using an Amicon® Ultra-15 centrifugal filter device with a 50 kDa nominal molecular weight cut off and the concentration determined using the Bradford method [58 (link)].

Whole bodies of larvae were finely chopped and mixed to obtain as much homogenous material as possible. After that, 0.2 g was taken to determine the concentration of thiobarbituric acid reactive substance (TBARS) as a standard marker for LPO (lipid peroxidation); the rest was used for determination of antioxidant parameters. Tadpoles were individually homogenized with an Ultra Turrax homogenizer T-18 (IKA-Werk, Staufen, Germany) at a 1:5 ratio in an ice cold 25 mM sucrose buffer (pH 7.4) containing 10 mM Tris-HCl and 5 mM EDTA. The homogenates were sonicated with an ultrasonic homogenizer (Sonopuls HD 2070, Bandelin electronic, Berlin, Germany) for 30 s at 10 kHz. A part of the sonicate was taken for measurement of total reduced glutathione (GSH) concentration, while the rest was centrifuged in an L7-55 ultracentrifuge (Beckman, Brea, CA, USA) at 100,000× g at 4 °C for 90 min [32 (link)]. The supernatants were used for measuring AOS parameters.

Lipid films were generated by drying lipid mixtures in chloroform under a stream of nitrogen at 50 °C and constant agitation at 400 rpm. Subsequently, residual chloroform was removed in an evacuated desiccator at RT for 1 h. Multilamellar liposomes were generated by the swelling method in liposome buffer (25 mM HEPES pH 7.0, 150 mM NaCl) at 1200 rpm shaking, 60 °C for 1 h. After sonication in a sonication bath for 20 min, the resulting multilamellar vesicles were snap-frozen in liquid nitrogen and stored at –80 °C. After thawing, the multilamellar vesicles were subjected to sonication using the sonotrode MS72 on a Bandelin Sonopuls HD 2070 with 50% power for 10 pulses of each 0.7 s. Liposomes were diluted (0.1 mM lipid) in liposome buffer and equilibrated in black 96-well plates in an Infinite 200 Pro (Tecan) plate reader at 30 °C. Background fluorescence emission spectra were recorded at 400–530 nm in 2 nm steps with 40 µs integration time, excitation at 375 nm and both bandwidths set to 10 nm. In total, 0.2 µM C-laurdan was added and incubated for 15 min before recording a fluorescence spectrum with the same settings. Generalized polarization was calculated as GP = (I1 − I2) / (I1 + I2) with I1 being the background-corrected signal sum between 400 and 460 nm and I2 the background-corrected signal sum between 470 and 530 nm.