JEV SA-14-14-2 and WT JEV AT31 were propagated in BHK-21 cells. H144A and Q258A mutant JEV virions were packaged using cDNA clones of JEV AT31 as described previously (39 (link)). PRRSV (HN07-1 strain) was propagated in MARC-145 cells. Collected JEV- and PRRSV-containing cell culture supernatants were centrifuged at 1,500 rpm and 4°C for 10 min and filtered with 0.2-μm-pore-size filters (Millipore) to remove cell debris. To evaluate the effect of ultracentrifugation on virus infectivity, part of the JEV SA-14-14-2 sample was further purified by ultracentrifugation (48 (link), 49 (link)). In brief, viruses were concentrated by centrifugation at 100,000 × g and 4°C in a Ty45 Ti rotor (Beckman) for 2 h, purified by gradient centrifugation on 10% to 35% potassium tartrate-glycerol (30%) at 125,000 × g in a SW32 Ti rotor for 2 h, and then desalted at 180,000 × g in the Ty45 Ti rotor for 1 h. IAV [A/chicken/Hubei/01-MA01/1999(H9N2) strain] was propagated in pathogen-free chicken eggs and purified by sucrose gradient ultracentrifugation as described previously (50 (link)). All the harvested viruses were subpackaged and stored at −80°C until use.
Ty45 ti rotor
Manufactured by Beckman Coulter
Sourced in United States
The Ty45 Ti rotor is a high-performance centrifuge rotor designed for Beckman Coulter ultracentrifuges. It is made of titanium and can achieve maximum speeds of up to 45,000 rpm, enabling the separation and isolation of a wide range of sample types, including proteins, nucleic acids, and cellular organelles.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
0.2 μm pore size filter, by Merck Group (1 mentions)
Plan apochromat 63 1.4 oil dic objective, by Zeiss (1 mentions)
Vivaspin column, by Sartorius (1 mentions)
Optima max xp, by Beckman Coulter (1 mentions)
Mls 50 rotor, by Beckman Coulter (1 mentions)
Centrifuge 5804 r, by Eppendorf (1 mentions)
Tla 55, by Beckman Coulter (1 mentions)
Nanodrop onec, by Thermo Fisher Scientific (1 mentions)
Biacore x100 instrument, by Cytiva (1 mentions)
Optima xpn 100, by Beckman Coulter (1 mentions)
G box chemi xx9, by Syngene (1 mentions)
2 protocols using ty45 ti rotor
1
Virus Propagation and Purification Protocols
2
Comprehensive Biophysical Characterization
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Centrifugation was performed with a 5804R Eppendorf Centrifuge equipped with an A-4-44 rotor, using 15 mL tubes. Ultracentrifugation for large volumes (up to 50 mL) was performed with an Optima XPN-100 equipped with a TY45 Ti rotor (Beckman Coulter, USA) while for small volumes (up to 1.5 mL) we used an Optima MAX-XP using a TLA-55 and a MLS-50 rotor (Beckman Coulter, USA). Images from western blot and gel fluorescence were acquired with a Syngene G : BOX Chemi XX9 (SYNGENE, UK) and were analyzed and quantified with the software Genesys and GeneTools (SYNGENE, UK). NanoDrop™ OneC (ThermoFisher, Rockford, USA) was used to characterize mCTX. A Zeiss LSM510 with a Plan-Apochromat 63×/1.4 Oil DIC objective (Germany) was used to perform fluorescence confocal microscopy. All the VivaSpin columns used in this work were purchased from Sartorius Stedim Lab Lid (Sperry Way, Stonehouse, UK). A NanoSight NS300 system was used to determine the size distribution and the particle concentration of REV samples (Malvern Technologies, Malvern, UK). A BIAcore X-100 instrument was used for SPR (Cytiva Life Science, Marlborough, MA, USA).
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