The fish were cleaned with tap water, and then the muscles were separated and rinsed with cold distilled water to remove the contaminants. The fish meat of T. ovatus was homogenized in a blender (Joyoung, JYL-Y910) for approximately 2 min. The homogenates were then hydrolyzed for 4 h (37 °C, pH 8.0) with trypsin at an enzyme to substrate ratio of 3000 U/g. After hydrolysis, the reaction was stopped by incubation in a boiling water bath for 10 min to inactivate the enzyme, followed by centrifugation at 8000 rpm (500 mL centrifuge tube, Avanti J-26S XP Centrifuge, Beckman Coulter, Inc. 250 S. Kraemer Blvd., Brea, CA 92821, USA.) for 20 min at 4 °C. A part of the supernatant was collected, concentrated and lyophilized to obtain T. ovatus hydrolysates (TOH). The other supernatant was collected and fractionated by ultrafiltration cassettes (Vivaflow 200: 10k and 3k molecular weight cutoff (MWCO) PES, Sartorius Stedim Biotech GmbH Göttingen, Germany) in an ice-water bath. The ultrafiltration fractions with molecular weights of 10 kDa–3 kDa (TOH-1) and <3 kDa (TOH-2) were collected.
Avanti j 26s xp centrifuge
Manufactured by Beckman Coulter
Sourced in United States
The Avanti J-26S XP Centrifuge is a high-performance benchtop centrifuge designed for a wide range of laboratory applications. It features a robust design and advanced technology to provide reliable and efficient sample separation.
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Lab products found in correlation
Optima xe 90 ultracentrifuge, by Beckman Coulter (1 mentions)
Sw28 swing bucket rotor, by Beckman Coulter (1 mentions)
Ja25.50 rotor, by Beckman Coulter (1 mentions)
Q700 sonicator, by Qsonica (1 mentions)
Ultracentrifuge, by Beckman Coulter (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (1 mentions)
8 protocols using avanti j 26s xp centrifuge
1
Enzymatic Hydrolysis of Trachurus Ovatus
2
Octopus Protein Hydrolysis Protocol
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The octopus homogenate was mixed with twofold (g/ml) distilled water and then hydrolyzed separately using alcalase (55°C, pH 8.0), neutrase (50°C, pH 6.5), and papain (50°C, pH 6.5) at 3000 U/g for 5 h. The reaction was stopped by boiling the samples in a water bath at 100°C for 10 min. The hydrolyzates were centrifuged at 30,000 g (Avanti J‐26S XP Centrifuge, BECKMAN COULTER, Inc.) for 30 min. The supernatant was collected, concentrated, and freeze dried, resulting in the octopus protein hydrolyzate ON, OA, and OP from neutrase, alcalase, and papain hydrolysis, respectively. The protein content of hydrolyzate powder was determined according to the AOAC method. The degree of hydrolysis (DH) was measured by the o‐phthaldialdehyde (OPA) method described by Nielsen et al. (2001 (link)).
3
Purification of Halobacterium salinarum PM
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Halobacterium salinarum S9 was grown as previously described, with no additional antibiotics necessary due to the culture conditions being too harsh for any contamination34 (link). The expression culture was grown at 38 °C with illumination in either an Erlenmeyer flask or a Winpact Bench-Top Fermenter (Major Science, Taiwan). The PM was purified from the bacterial pellet using a standard sucrose gradient procedure, in an Optima XE-90 Ultracentrifuge with the SW28 swing bucket rotor (Beckman Coulter, USA) spinning at 87300 × g for 22 h (Supplementary Figure S1 ). After the overnight centrifugation, the PM was extracted and underwent a fast centrifugation in an Avanti J-26S XP Centrifuge with JA25.50 rotor (Beckman Coulter, USA) at 27216 × g for 1 h to remove any residual sucrose. The PM pellet was then resuspended in water and gently sonicated using a Q700 sonicator (QSonica, USA). The concentration of PM was subsequently estimated with steady-state spectroscopy, using the absorption at 568 nm and the extinction coefficient of bR ε568 = 62700 M−1 cm−135 (link). The PM was then stored at 4 °C.
4
Isolation of Milk-Derived Extracellular Vesicles
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Milk derived EVs (mEVs) were isolated using ultracentrifugation‐based method with some modifications (Somiya et al., 2018 (link); Yamauchi et al., 2019 (link)). Briefly, three litres of fresh unpasteurised milk was collected from a local dairy farm. The collected milk was aliquoted (500 mL) at −80°C and used within 3 days. The mEV samples were isolated by differential centrifugation. Firstly, raw milk was centrifuged at 2000 × g for 5 min at 4°C to remove milk fat globules and cells, and centrifuged again at 10,000 × g for 30 min to remove the fat residues and cellular debris. Then the supernatant was acidized to pH = 4.6 with acetic acid to aggregate the caseins, followed by 10 min incubation and 15 min centrifugation at 10,000 × g at 4°C. The supernatant was sequentially filtrated through 1, 0.45, and 0.22 μm polyethersulfone (PES) membranes to remove large particles, and centrifuged at 100,000 × g for 70 min to pellet EVs. Finally, the mEVs pellets were resuspended in PBS or ammonium sulfate for further experiments. A Beckman Coulter Avanti J‐26S XP centrifuge and a Beckman Coulter ultracentrifuge were used for the centrifugation throughout present study.
5
Enzymatic Hydrolysis of Chicken By-Products
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15 kg (wet weight) minced chicken by-products were mixed with 15 L of water in 30 L Einar hydrolysis reactors (Belach Bioteknik, Skogås, Stockholm, Sweden), resulting in a dry-matter concentration of 15%. The enzymatic hydrolysis of the chicken by-products was carried out using 0.5% (weight of the enzyme powder/weight of wet chicken by-products) papain from Carica papaya, (≥ 3 U/mg; Merck, Darmstadt, Germany) at 60 °C and 50 rpm without pH adjustment and using slow heating to 60 °C, as described previously [20 (link)]. The hydrolysates were removed from the hydrolysis tanks after 2 h and were filtered through a sieve of 0.85 mm Ø in order to remove insoluble particles. Subsequently, the hydrolysates were cooled down to 4 °C and stored overnight, which led to accumulation of lipids on the top of the hydrolysate. The liquid fraction was centrifuged in a Beckman Coulter Avanti J-26S XP centrifuge (Indianapolis, Indiana, USA) at 4 °C and 10.000 g for 10 min. Finally, the chicken by-products hydrolysates (CH) were filtered using a sieve of 75 µm Ø and stored at − 20 °C until use. Due to the large hydrolysis volume (15 kg raw material and 15 kg water), the inactivation of proteolytic enzymes was not carried out directly after hydrolysis but by autoclaving of specific aliquot volumes used when preparing fermentation media.
6
Chloroform-based PHBV Extraction Protocol
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The unpurified PHBV was extracted using the chloroform-based extraction method reported previously 37 . For this, the MBW derived PHBV was dissolved in chloroform at 5 wt.% and the mixture was stirred for 24 h at 50 ºC to degrade the non-PHA cellular material. Next, the solution was transferred to centrifugation tubes in which distilled water was added at 50 wt.%. After shaking the tubes manually, these were centrifuged for 5 min at 4000 rpm in an Avanti J-26S XP Centrifuge from Beckman Coulter, Inc.
(Brea, CA, USA). Finally, the PHBV suspension was recovered from the bottom of the tubes with a pipette and transferred to beakers, leaving them in the extractor hood until the solvent was completely evaporated.
(Brea, CA, USA). Finally, the PHBV suspension was recovered from the bottom of the tubes with a pipette and transferred to beakers, leaving them in the extractor hood until the solvent was completely evaporated.
7
Isolation and Characterization of Adipose-Derived sEVs
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sEVs used in this study were isolated using the Total Exosome Isolation (TEI) reagent with minor modification. Briefly, 5 g of adipose tissue were collected from 8-week-old male C57BL/6 mice, washed extensively with sterile phosphate-buffered saline (PBS) to remove the debris and red blood cells. The tissue was cut into small pieces (1–2 mm3) under aseptic condition and then treated with 10 ml 0.075% collagenase (type I) for 30 min at 37 °C. The digested adipose tissue was centrifuged at 300g for 10 min and the supernatant (SN-AT) was collected, filtered (0.22 μm filter) to remove the debris of cells. Then the supernatant was concentrated with Amicon® Ultra-15 Centrifugal Filter Units (10,000 Mw cut off the membrane, Millipore, USA) at the speed of 5000g for 30 min (4 °C, Beckman Avanti J-26S XP centrifuge, JS5.30). The concentrated medium was mixed with 0.5 volume of Total Exosome Isolation™ reagent (Life Technologies, USA), incubated overnight at 4 °C and spun down for 1 h at 10,000 g at 4 °C. The pellet was re-suspended in 100 μL and used for cell treatment or injection in vivo.
8
Measuring Milk Sedimentation by Centrifugation
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Only sedimentation was observed in samples during storage period. Sedimentation was measured by a centrifugation method as previously described (Boumpa, Tsioulpas, Grandison, & Lewis, 2008) with slight modifications. The milk packs were well shaken and approximately 40 g was accurately weighed in a centrifuge tube and centrifuged at 4000 rpm (2760 g) for 15 min (Beckman Coulter Avanti J-26S XP Centrifuge, CA, USA). The sediment was then oven-dried at 50 °C for 48 hours to a constant weight which was then expressed as the amount of sediment (mg/100g of milk).
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