The 60 × 106 human melanoma cells were grown in serum-free MIM for 48 h. Collected supernatant fractions were filtered with a 0.2 µm filter (Sarstedt, Nümbrecht, Germany). Vesicles were pelleted by ultracentrifugation at 34800 rpm for 70 min at 4 °C (Beckman Coulter 55.2 Ti rotor, Krefeld, Germany). The pellet, containing vesicles, was resuspended in 100 µl PBS. Vesicle concentration and size were measured using the Nanosight technology equipped with a blue laser (405 nm) for real-time characterization of the vesicles.
Rotor 55.2 ti
Manufactured by Beckman Coulter
Sourced in United States
The Rotor 55.2 Ti is a high-performance centrifuge rotor designed for use with Beckman Coulter's ultracentrifuge systems. It is constructed from titanium for enhanced durability and chemical resistance. The rotor can accommodate up to 6 sample tubes and is capable of reaching a maximum speed of 55,000 rpm, enabling efficient separation of complex biological samples.
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7 protocols using rotor 55.2 ti
1
Isolation and Characterization of Melanoma-Derived Extracellular Vesicles
2
Cell Lysis and Protein Extraction
3
Fractionation of Microsomal Membranes by FFZE
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Microsomal membranes were fractionated by FFZE using the BD FFE system (BD Proteomics, Germany), as previously described (Barkla et al., 2007; Barkla, 2018) (link). Before fractionation, microsomes were diluted 1:1 (v/v) in a separation medium composed of 250 mM sucrose, 2 mM KCl, 10 mM TEA, 10 mM acetic acid pH 7.4 and centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant was collected, adding 25 µL of 6 mM MgSO 4 , and 25 µL of 3 mM ATP. The sample (3 mg/mL) was injected continuously via a peristaltic pump at a rate of 1.2 mL/h using the anodic sample inlet. Membrane fractions were collected continually in 96 deep-well microtiter plates (4 mL/well; Sunergia Medical, VA). Fractions from sequential runs were pooled, and membranes were concentrated by centrifugation in a Beckman 55.2 Ti rotor in an L8-M ultracentrifuge at 100,000 × g av for 50 min at 4°C. Membrane pellets were resuspended in 50-100 µL of suspension buffer containing 250 mM mannitol, 10% glycerol (w/v), 10 mM Tris/MES pH 8, and 2 mM DTT and frozen in liquid N 2 for storage at -80°C.
4
Isolation of Extracellular Vesicles
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In order to avoid the presence of RS-visible isolation reagent residues in the EV suspension, the vesicles were isolated from cell-conditioned medium (CM) by means of differential centrifugation, as previously described43 . Briefly, after 72 hours of starvation, the medium conditioned from approximately 6 × 106 cells was centrifuged at 800 g for 10 min to remove non-adherent cells and then at 2,500 g for 15 min to remove potential apoptotic bodies. CM was then ultracentrifuged for 70 min at 100,000 g (L7–65; Rotor 55.2 Ti; Beckman Coulter, Brea, CA, USA) at 4 °C, and the pellet was re- suspended in sterile saline solution and ultracentrifuged again. The collected EV suspension (approximately 500 µl) was kept at 4 °C before making Raman and TEM analyses, and then frozen.
5
Extracellular Vesicle Isolation Protocol
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Extracellular vesicles were isolated from cell-conditioned medium using differential centrifugation, as previously described [49 (link),50 (link)]. In brief, after 72 h of starvation, the conditioned medium from approximately 15 × 106 cells was centrifuged for 15 min at 2500× g, 4 °C, and then ultra-centrifuged for 70 min at 100,000× g (L7–65; Rotor 55.2 Ti; Beckman Coulter, Brea, CA, USA) at 4 °C. Pellet was resuspended in sterile PBS and ultra-centrifuged again under the same conditions. The resulting EV pellet was kept at −20 °C for mass spectrometry analysis.
6
Isolation and Characterization of Cell Secretome
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ASC, BMSC, and DF from IV to XI passage at 90% of confluence were incubated in starving conditions for 72 hours (the absence of FBS). No signals of cell suffering were ever observed. The medium was collected and centrifuged at 2500g for 15 minutes at 4°C to remove dead cells, large apoptotic bodies, and debris. The supernatants were split in half to obtain paired CM and EV samples, while donor cells were counted in order to correlate cell number to the final products (CM or EV). An aliquot of the CM was concentrated about 40-50 times by centrifuging at 4000g for 90 minutes at 4°C in AmiconUltra-15 Centrifugal Filter Devices with 3 kDa molecular weight cutoff (Merck Millipore, Burlington, MA, USA). This procedure allows the retention of the vesicular component of cell secretome, as previously demonstrated in the studies of Carlomagno et al., Niada et al., and Giannasi et al.13 ,14 (link),18 In parallel, EV were isolated starting from CM through differential centrifugation at 100 000g (L7-65; Rotor 55.2 Ti; Beckman Coulter, Brea, CA, USA), 4°C for 70 minutes).14 (link),21 (link) The resulting CM and EV were kept at −80°C until use.
7
Prophage Induction in Komagataeibacter
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Prophages were induced in the log-phase growing culture Komagataeibacter intermedius IMBG180 in HS medium with polychromatic UV. Medium pressure mercury vapor lamp (polychromatic light, 200-600 nm wavelengths) (Jelight Company, Inc) was used to irradiate the liquid cultures in open Petri dishes with a dose 10 J/m . After appropriate treatment, the culture was centrifuged at 10,000 rpm for 20 min at 4 °C. The supernatant was collected and further ultra-centrifuged at 100,000 g for 1 h at 4 °C (Beckman Instruments Inc., L8M, rotor 55.2 Ti). The resulting pellet was re-suspended in sterile 0.9 % saline.
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