Sonic ruptor 400

Hemi-sected transgenic mouse brains were stored at −80 °C prior to homogenization. Frozen brains were placed onto a chilled metal block on dry ice and were transected coronally at the level of the midbrain to separate caudal and rostral brain sections (see also Fig. 15). Sections were placed in 1 mL PBS prepared with protease inhibitor tablet (Roche) and were sonicated using a probe sonicator (Omni Sonic Ruptor 400) at 30% pulse/30% power, 20 times, 10-s cycles. The resulting homogenates were centrifuged at 10,000 xg for 15 min at 4 °C. and supernatants used for subsequent steps [27 (link)]. Clone 1, Clone 9, and Clone 10 lysates were prepared as described previously [27 (link)]. Cell pellets were lysed in 0.1% Triton-X/PBS solution plus protease inhibitor cocktail (“Complete”, Roche) and were clarified with sequential five-minute 500 xg and 1000 xg spins. Protein concentrations in brain homogenates and cell lysates were measured by Bradford assay (Bio-Rad) and were normalized to 5 μg/μL and stored at −80 °C until use.

HUVECs and HMVEC-dLyAd cells were subcultivated using trypsin, then centrifuged and counted. To obtain a homogenate containing an equivalent cell number, cells were centrifuged again and re-suspended in an appropriate volume of cold RIPA buffer containing a protease inhibitor cocktail. Cells were disrupted using a probe sonicator on ice (Sonic Ruptor 400, OMNI International). Insoluble cellular components were removed by centrifugation at 15,000 RPM for 10 min at 4 °C. The protein content of the supernatant was determined according to the Bradford protein assay using BSA as the standard [33 (link)]. The enzymatic activity was tested on 5 μg of protein using L-Met-AMC (Santa Cruz Biotechnology) as a substrate. The reaction was performed in an assay buffer (pH 7.5) containing 50 mM HEPES, 0.1 mM CoCl2, 100 mM NaCl and 1 mg/mL PEG 6000, in a final volume of 100 μL. Fluorescence was measured every 20 sec for 1 h at 25 °C using a plate reader (Synergy HT Multi-Mode Microplate Reader, BioTek), n = 2.

Each lyophilized plant and cyanobacterial sample was mixed with 4 mL of 80% acetone and homogenized on an Omni Sonic Ruptor 400 homogenizer (USA) on ice (3/8” processing tip, gradually increasing the power up to 400 watts, 10 s homogenization). Verification of complete cell lysis was performed microscopically. After the samples were centrifuged for 5 min at 10,000× g, 200 µL of the supernatant was transferred to a 96-well plate, and the absorbance at 470, 646, and 665 nm wavelengths was measured on a BioTek Synergy H1 microplate reader. Photosynthetic pigments content was calculated using the equations developed by Wellburn [24 (link)]. A total of 1 mL of sample was collected for the analysis of cyanotoxins concentration.

One hundred grams of DPP grains were extracted using ethanol 80% for 24h then homogenized using ‎‎300VT Ultrasonic Homogenizer (BioLogics, Inc. Manassas, VG, USA) at ‎temperature below 25°C [19 (link)]. The extracts were filtered and concentrated using rotary evaporator at 45°C. The extract was evaporated under reduced pressure, ‎lyophilized to give a yellow semisolid residue (free extract) (25g) and stored in dark container at 4°C ‎until use.
For encapsulation, Sodium caseinate (NaCas) was hydrated to 5% w/v in deionized water overnight at room temperature (21°C). Soy lecithin 0.5% w/v dissolved in (10 mM sodium phosphate, pH 7). lecithin was mixed with the NaCas solution in ratio 1:1 by gentle magnetic stirring for 1 h. DPP extract was then added to the wall material 10%, and the nanocapsules solution was formed using an Ultra-Turrax homogenizer T18 basic (IKA, Wilmington, USA), operating at a speed of 18,000 rpm for 5 min, ultrasonicated at 160 W power, 20 kHz frequency and with 50% pulse (Sonic Ruptor 400, OMNI International the Homogenizer).