Method B was based on ultracentrifugation, which is frequently used to concentrate viruses from wastewater (Fumian et al., 2010 (link)). In this study, we used the protocol published by Ahmed et al., (2020) (link) with some modifications. The wastewater/ESRM sample (20 ml) were poured to polycarbonate ultracentrifuge bottles (# 355,618, Beckman Coulter, Indianapolis, Indiana, USA) and subjected to ultracentrifugation at 100000g for 1 h at 4 °C (Type 70 Ti rotor, Beckman Coulter, # 337,922) in an Optima XPN-90 Ultracentrifuge (Beckman Coulter, # A94468). The supernatant was removed, and the pellet was resuspended in 3.5 mL of 0.25 N glycine buffer (pH 9.5). Then, the sample was incubated on ice for 30 min and 3 mL of 2x phosphate buffered saline (PBS; pH 7.2) for neutralization was added. The sample was centrifuged at 12000g for 15 min at 4 °C. After that, the virus particles were recovered by ultracentrifugation again in polycarbonate ultracentrifuge bottles (Beckman Coulter, # 355,603) at 100000g for 1 h at 4 °C using a Type 90 Ti rotor (Beckman Coulter, # 355,530) in an Optima XPN-90 Ultracentrifuge. The pellet was resuspended in 400 μl of PBS (pH 7.2) and transferred to a microtube (1.5 mL) for RNA extraction. RNA was extracted using the two kits (NucleoSpin RNA Virus Kit, AllPrep PowerViral DNA/RNA Kit) as described above.
Optima xpn 90 ultracentrifuge
Manufactured by Beckman Coulter
Sourced in United States
The Optima XPN-90 Ultracentrifuge is a high-performance laboratory instrument designed for the separation and analysis of macromolecules and cellular components. It features a maximum rotor speed of 90,000 rpm and a maximum relative centrifugal force of 803,000 x g, enabling efficient separation of materials based on their size, shape, and density.
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Lab products found in correlation
5 protocols using optima xpn 90 ultracentrifuge
1
Virus Concentration from Wastewater
2
Preparation and Purification of Membrane Vesicles
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Membrane vesicles were prepared as described previously with a slight modification (20 (link)). Wild-type and codY mutant cells of USA300 LAC or UAMS-1 were grown for 5 h in TSB. Cell pellets were frozen and resuspended in TSM (50 mM Tris [pH 8.0], 0.5 M sucrose, 10 mM MgCl2) containing 40 μg mL−1 lysostaphin and incubated for 20 min at 37°C. An additional 2 mL of membrane lysis buffer (100 mM Tris [pH 8.0], 100 mM NaCl, 10 mM MgCl2) was added, and cells were lysed by sonication (Fisherbrand model 120 sonic dismembrator). A 100-μL sample of whole-cell lysate was saved for Western blot analysis, and the remaining lysate including the membrane fraction was centrifuged at 7,000 × g for 5 min. The membrane fraction was recovered via ultracentrifugation at 82,700 × g for 30 min (Optima XPN-90 ultracentrifuge; Beckman). The pellet was washed and pelleted through three additional rounds of ultracentrifugation (82,700 × g, 30 min each round), first in 20 mM Tris (pH 8.0) plus 2 M KCl, then in 20 mM Tris (pH 8.0) plus 5 mM EDTA, and then in 20 mM Tris (pH 8.0). Finally, the pellet was resuspended in TKMG buffer (50 mM Tris [pH 8.0], 50 mM KCl, 1 mM MgCl2, 25% [vol/vol] glycerol) and stored at −80°C until use.
3
Lentiviral Vector Production Protocol
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The lentivirus was produced by transfecting GFP-Luc or CRISPR knockout library plasmids (10 µg) into HEK293FT cells in a 15-cm dish together with the viral packing plasmid Delta8.2 (7.5 µg) and the envelope plasmid VSVG (5 µg). The supernatant containing viral particles was harvested two times at 48 hours and 60 hours. All the supernatants were filtered through 0.45 μm polyethersulfone (PES) filters and pooled together to be concentrated at 70,000 g for 1.5 hours at 4 °C by Optima XPN-90 Ultracentrifuge (Beckman). The pellet was washed once with PBS, and the virus was resuspend again in PBS. The suspension was divided into aliquots, which were stored at -80 °C for less than 1 month.
4
Amniotic Fluid-Derived Extracellular Vesicle Isolation
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Full-term human amniotic fluid was collected in the operating room by aspiration during planned cesarean sections and shipped to the manufacturing faculty on ice. Upon processing, raw AF was centrifuged at 500g for 10 minutes. The supernatant was collected and centrifuged at 2000g for 20 minutes. The supernatant was then filtered through a.22µm filter to remove all large cell debris and large proteins. The processed product resulted in acAF comprising acellular extracellular components such as EVs and soluble proteins.
A starting volume of 16ml acAF was used for AF-EV preparations. To create the AF-EV preparation, acAF was further centrifuged at 100,000g for 3 hours at 4°C using an Optima XPN-90 Ultracentrifuge (Beckman Coulter) with a type 50.4 Ti fixed-angle titanium rotor (Beckman Coulter). The supernatant was removed, and the pellet was washed by resuspending in sterile DPBS and centrifuging a second time at 100,000g for 2 hours at 4°C. The final EV pellet was resuspended in 500µl DPBS and stored at -80°C until use.
EV-depleted supernatant was prepared by collecting acAF supernatant from the first centrifugation of AF-EV preparations, and centrifuging it at 100,000g for 16 hours. The EV-depleted supernatant was then collected.
A starting volume of 16ml acAF was used for AF-EV preparations. To create the AF-EV preparation, acAF was further centrifuged at 100,000g for 3 hours at 4°C using an Optima XPN-90 Ultracentrifuge (Beckman Coulter) with a type 50.4 Ti fixed-angle titanium rotor (Beckman Coulter). The supernatant was removed, and the pellet was washed by resuspending in sterile DPBS and centrifuging a second time at 100,000g for 2 hours at 4°C. The final EV pellet was resuspended in 500µl DPBS and stored at -80°C until use.
EV-depleted supernatant was prepared by collecting acAF supernatant from the first centrifugation of AF-EV preparations, and centrifuging it at 100,000g for 16 hours. The EV-depleted supernatant was then collected.
5
Isolation of Synaptic Mitochondria from Brain Tissue
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