Sorvall mtx 150 micro ultracentrifuge

Both microvesicles and exosomes were isolated from LVL through serial centrifugation (Supplementary Fig. 3), according to Thery et al. [58 ], and Gurunathan et al. [46 ], with some modifications. In brief, LVL from each mare was centrifuged at 2000 xg (Sigma 3-18KS, UK) for 20 min at 4 °C, in order to remove blood and cellular debris. Supernatant then was transferred into new tubes, and centrifuged at 25,000 xg (Thermo Scientific™ Sorvall™ MTX 150 micro-ultracentrifuge, USA) using a fixed angle rotor for 30 min at 4 °C, to get a microvesicles pellet. The microvesicles pellet was suspended in phosphate buffered saline (1x PBS), and then kept at − 80 °C. Afterwards, supernatant was ultracentrifuged at 120,000 xg (Thermo Scientific™ Sorvall™ MTX 150 micro-ultracentrifuge, USA), using a fixed angle rotor, for 190 min at 4 °C to get an exosomes pellet. Then the pellet was suspended in 1x PBS and kept at − 80 °C, for further analysis.
Also, microvesicles and exosomes were isolated from serum using an ExoQuick® Ultra EV kit (System Biosciences, USA). Serum from each mare was thawed on ice, and the extraction procedure was performed according to the manufacturer’s protocol. Afterwards, the isolated EVs were kept at − 80 °C, until further analysis.

To measure the concentration of silver species in the dissolution products used for antimicrobial studies 70S28C2A fibres were soaked in RPMI 1640 (with a ratio of 10 mg/ml) for 3 days and then filter-sterilised using a 0.22 μm filter and incubated at 37 °C. At time points t = 0, 1, 3 & 7 days, 2 ml of the dissolution products were ultracentrifuged, using the Sorvall MTX 150 Micro-ultracentrifuge from Thermo Fisher Scientific, at 35,000 rpm for 30 min. This was to separate the silver ions from silver nanoparticles and silver chloride. The supernatant was mixed with 2 ml of RO water and 2 ml of 32% ammonium hydroxide. This was diluted 1:100 in RO water. To collect the silver nanoparticles, the pellet formed from ultracentrifugation was resuspended in 5 ml of 10% nitric acid and was then centrifuged using the Hettich Zentrifugen Universal 320r at 4000 rpm for 1 h to dissolve the nanoparticles in the supernatant. This was then diluted 1:100 in RO water. The silver ion concentration and nanoparticle concentration were measured using the PerkinElmer Nexion 300X model for induced coupled plasma mass spectroscopy (ICP-ms). Three experimental repeats were performed.

Total protein extracts were prepared in 100 mM NaCl; 50 mM Tris–HCl pH 7.5; 0.5% (v/v) Triton X-100; 1 mM DTT; 1× Complete Protease Inhibitor Cocktail Tablet (Roche), and spectrophotometrically quantified using ROTIQUANT (Carl Roth). A 10–20 µg aliquot of protein extract was loaded onto an SDS-polyacrylamide gel, blotted on nitrocellulose membranes, probed with antibodies listed in Supplementary Table S3 at JXB online, and developed with the SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) in a Luminescent Image Analyzer LAS4000 System (Fujifilm). Biochemical fractionation was conducted as previously described (Garcia-Molina et al., 2014 (link)) from 7-day-old CaMV35S:HA-LSU1/2 seedlings in a Sorvall™ MTX 150 Micro-Ultracentrifuge (Thermo Scientific) with a S55A2 rotor. Fractions were assayed by western blots with antibodies anti-H3, anti-UGPase, anti-V-ATPase, and anti-haemagglutinin (HA) (Supplementary Table S3).