The viruses in the allantoic fluid were first inactivated using 0.05% BPL as described previously2 . To concentrate the viruses, allantoic fluids were clarified by centrifugation at 3441 × g at 4 °C for 30 min using a Sorvall Legend RT Plus Refrigerated Benchtop Centrifuge (Thermo Fisher Scientific). Clarified allantoic fluids were laid on top of a 20% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4). Ultracentrifugation in a Beckman L7-65 ultracentrifuge at 112,499 × g for 2 hours at 4 °C using a Beckman SW28 rotor (Beckman Coulter) was performed to pellet the viruses through the sucrose cushion while soluble egg proteins were removed. The virus pellets were resuspended in PBS (pH 7.4). The total protein content was determined using the bicinchoninic acid assay (Thermo Fisher Scientific).
L7 65
Manufactured by Beckman Coulter
Sourced in United States
The L7-65 is a high-speed centrifuge that provides reliable and efficient sample processing. It is designed for a wide range of applications in clinical and research laboratories. The L7-65 features a robust and compact design, ensuring consistent performance and ease of use.
Automatically generated - may contain errors
Lab products found in correlation
Beckman sw28 rotor, by Beckman Coulter (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Sorvall legend rt plus refrigerated benchtop centrifuge, by Thermo Fisher Scientific (1 mentions)
Polycarbonate centrifuge bottles, by Beckman Coulter (1 mentions)
Polypropylene tubes, by Beckman Coulter (1 mentions)
Rotor 70.1ti, by Beckman Coulter (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)
Sw28 rotor, by Beckman Coulter (1 mentions)
Sw40 swing out rotor, by Beckman Coulter (1 mentions)
Sw41ti, by Beckman Coulter (1 mentions)
Rotor 55.2 ti, by Beckman Coulter (1 mentions)
Amicon ultra 15 centrifugal filter device, by Merck Group (1 mentions)
8 protocols using l7 65
1
Virus Concentration and Purification from Allantoic Fluid
2
Virus Isolation and Protein Extraction
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Fifty millilitres of the virus supernatant cultured in Vero cells were collected, and cell debris was removed by centrifugation at 1000×g for 15 min at 4 °C. Viruses in the supernatant were concentrated by ultracentrifugation in polycarbonate centrifuge bottles (no. 355603, Beckman Coulter) using a Beckman L7–65 ultracentrifuge (rotor 70.1 Ti) set at 35,000 rpm for 1.5 h at 4 °C. After ultracentrifugation, pellets containing viruses were processed for mass spectrometric analysis via re-suspension in lysis buffer (1% NaCl, 1% SDS and 1% Triton-X) to produce a virus protein lysate.
3
Isolation and Characterization of Small Extracellular Vesicles
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One milliliter of cell culture supernatant, 500 μl of 1:2 diluted serum (with phosphate buffered saline, PBS, Sigma-Aldrich, Darmstadt, GER) or synovial fluid from RA patients were centrifuged at 2,000 xg at room temperature for 20 min. The synovial fluid was pre-treated with hyaluronidase (1,500 U/ml; Sigma-Aldrich, Darmstadt, GER) for 15 min at 37°C, before sEV were isolated. The supernatant was centrifuged at 21,000 xg in 1.5 ml polypropylene tubes (Beckman Coulter, Brea, USA) at 4°C for 60 min in a L7-65 ultracentrifuge using rotor 70.1.Ti (Beckman Coulter, Brea, USA) to remove large membrane vesicles. The supernatant was transferred in a new polypropylene tube and centrifuged at 100,000 xg at 4°C for 60 min. The supernatant was discarded. The remaining pellet was resuspended in PBS. Quantity of purified sEV was determined on protein level via UV-Vis spectroscopy to ensure treatment with equal amounts of sEV between sEV-treatment experiments. SEV were used directly or stored at 4°C for not longer than 1 week.
4
Influenza Virus Purification and Quantification
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Influenza viruses were grown in 10-day old embryonated chicken eggs at 37 °C for two days, and were then cooled at 4 °C overnight. Allantoic fluids were collected and clarified by low speed centrifugation. Viruses in the clarified allantoic fluids were inactivated with 0.03% methanol-free formaldehyde for 48 h at 4 °C with rocking. Viruses were then pelleted through a 30% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) by centrifugation in a Beckman L7-65 ultracentrifuge at 25,000 rpm for 2 h at 4 °C using a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Pellets were collected in PBS (pH 7.4), and protein content was quantified using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).
5
Purification of Viruses from Allantoic Fluid
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Viruses were grown in 10-day old eggs and allantoic fluid was harvested and clarified by centrifugation at 290 x g using a benchtop centrifuge (Eppendorf) at 4 °C for 5 min. The clarified supernatant was concentrated using a 30% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 8) by centrifugation in a Beckman L7-65 ultracentrifuge at 87041.1 x g for 2 h at 4 °C using a Beckman SW28 rotor. Supernatant was aspirated and pellets were resuspended in PBS and stored at −80 °C in small aliquots.
6
Extracellular Vesicle Isolation Protocol
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10 mL of freshly harvested conditioned media were centrifuged at 110,000 × g for 2 h at 4°C in an Optima L7-65 ultracentrifuge using a SW40 swing-out rotor (k factor 299, Beckman Coulter). Obtained pellets were re-suspended in 1 mL phosphate-buffered saline (PBS; Life Technologies). Obtained EVs were either processed immediately, kept on −20°C for short-term or −80°C for long-term storage.
7
Isolation and Characterization of Osteoblast-Derived Extracellular Vesicles
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Osteoblast derived EVs (OB-EVs) were isolated according to Ucci et al. (20 (link)) and Loftus et al. (21 (link)). Briefly, upon reaching 80% confluence, mouse primary osteoblasts were washed in DPBS and starved in serum-free DMEM to prevent contamination from FBS-EVs. After 24 h, the conditioned medium (CM) was collected and sequentially centrifuged at 300 x g, 4°C for 5 min to remove dead cells and at 5,000 x g, 4°C for 25 min to remove membrane debris. The supernatant was collected and transferred to a Beckman L7-65 ultracentrifuge in a Beckman SW41-Ti or SW28 rotor and centrifuged at 100,000 x g, at 9°C for 70 min. Supernatant was discarded, while the pellet, containing EVs, was resuspended in DMEM for cell treatments. To quantify OB-EVs, they were subjected to nanoparticle tracking analysis (see Supplementary Figure 1A
8
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.
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