Gs 15r centrifuge

EVs were isolated from the plasma of fresh blood samples or blood previously stored at 4°C for 21 days. First, the samples were centrifuged (2,000 g for 10 minutes at 4°C) for the removal of cell debris (Eppendorf 5415R, Hamburg, Germany). Thereafter, the obtained supernatant was centrifuged again (20,000 g, for 70 minutes, at 4°C) (Beckman, GS-15R Centrifuge). A control sample containing 1 μm microspheres (Life Technologies, Carlsbad, CA, USA) was used to accurately define the FSC/SSC profile containing the MPs (<1 μm events) with adequate accuracy on the flow cytometer. Samples were labeled with the CD235a-FITC antibody (anti-glycophorin) at 1:100 concentration (clone 11E4B-7-6, IM2212U, Beckman Coulter, France) and 1 μm microspheres (Invitrogen, Waltham, USA) were labeled at 1: 100 concentrations with the FITC Mouse IgG1 isotypic control (clone MOPC 21, BD Biosciences, New Jersey, USA). A total of 100 μL/sample was acquired on the Accuri c6™ cytometer (BD Biosciences, CA, USA). RBC-EVs were also resuspended in sterile PBS and stored at -20°C until use.

The tissues were moved from −80°C and heated in 95°C acetic acid (1M) for 5 min, then placed on ice and homogenized by sonication using a Branson Sonifier (Danbury, CT, USA). The homogenates were centrifuged for 15 min at 4°C, 12,000 × g in a Beckman GS-15R centrifuge (Fullerton, CA, USA) and supernatants were purified by cation exchange chromatography procedure (24 (link)). The purified samples were dried in a vacuum centrifuge and stored at −20°C.

According to a previously described method (44 (link)), 500 mL of phage lysate was precipitated with 10% (wt/vol) PEG 6000 (Sigma-Aldrich) and 0.5 M NaCl and incubated overnight at 4°C with agitation. Bacterial cells were removed by centrifugation at 4,400 × g (Beckman Coulter, Aventi JE Centrifuge) for 30 min at 4°C. The pellet was resuspended in TM buffer. PEG was removed twice by adding an equal volume of chloroform, mixed by gentle inversion for 30 s, and centrifuged at 3,800 × g (Beckman, GS-15R Centrifuge) for 10 min. Then, 0.75 g of cesium chloride (CsCl) was added per mL of the aqueous phase containing phage particles (45 (link)). The solution was centrifuged at 31,000 rpm (Beckman Coulter, Optima L80 XP Ultracentrifuge) for 48 h at 10°C in a SW 41 Ti rotor. The phage band was recovered by piercing through the ultracentrifuge tube with a sterile 25-G needle (Terumo Corporation). To remove CsCl, the sample was transferred in a dialysis cassette G₂ (Thermo Fisher Scientific) and dialyzed against 1.5 L of TM buffer at 4°C overnight.

This property was determined according to AACC method 88-04 [7 ]. Approximate water absorption capacity was first determined by weighing out 0.1 g (d.b.) of sample, adding water until saturation (approximately 5 mL), and centrifuging at 2000 ×g for 10 min in a Beckman GS-15R centrifuge. Excess water was discarded and the residue was weighed. Approximate water absorption capacity was calculated by dividing the increase in sample weight (g) by the quantity of water needed to complete original sample weight to 15 g. Water absorption capacity (WA_bC) was then determined by placing samples in four tubes, adding different quantities of water to bracket the measurement (1.5 and 0.5 mL water above original weight and 1.5 and 0.5 mL water below; one in each tube), agitating vigorously in a vortex for 2 min, and centrifuging at 2000 ×g for 10 min in a Beckman GS-15R centrifuge. The supernatant was discarded and the residue was weighed. Average water absorbed was calculated and the WA_bC was calculated, expressed as g water absorbed per g of sample.

Mucoadhesive properties were determined through turbidimetric analysis of a suspension containing mucin and BS [30 (link)]. Mucin (Type II: crude, from porcine stomach, Sigma-Aldrich) was dispersed in water (0.08% w/v) and after 24 h the dispersion was centrifuged at 7500 rpm (GS-15R Centrifuge, Beckman Coulter, Milan, Italy) for 20 min in order to discharge the excess of mucin. Subsequently, the isolated mucin supernatant and a BS solution (1.25 mg/mL) were mixed (1:3 v/v) for 3 h. The turbidity of the mixture was measured at 650 nm (UV–Visible Spectrophotometer, Shimadzu Corporation, Australia) and compared to the controls (mucin supernatant and BS solution without mucin).