The two viral samples obtained by iron chloride and PEG 8000 precipitation were pooled together and bacteriophages were further concentrated by ultracentrifugation (Optima XE90, Beckman-Coulter, rotor type 90 Ti, Beckman-Coulter) at 77,000 rcf for 1 h at 4°C (Szpara et al., 2011 (link)). A P1 reference bacteriophage NC_005856.1 was used as a positive control for DNA extraction. The pellet was resuspended in 1 ml of supernatant and the total DNA was extracted according to the protocol published by Thurber et al. (2009 (link)). Using the Qubit dsDNA HS assay kit (Life), the extracted DNA was quantified at 0.2 ng DNA.
Type 90 ti rotor
Manufactured by Beckman Coulter
Sourced in United States
The Type 90 Ti Rotor is a high-performance centrifuge rotor designed for use with Beckman Coulter ultracentrifuges. It is constructed from titanium, which provides exceptional strength and durability. The rotor has a maximum speed of 90,000 rpm and can accommodate a variety of sample tubes, enabling efficient separation and purification of a wide range of biological molecules and particles.
Automatically generated - may contain errors
Lab products found in correlation
Qev size exclusion column, by Izon Science (3 mentions)
Optima xe 90, by Beckman Coulter (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
0.22 μm polyethersulfone membrane, by Merck Group (2 mentions)
Pi88700, by Thermo Fisher Scientific (2 mentions)
Sybr green qpcr super mix udg with rox, by Thermo Fisher Scientific (1 mentions)
Applied biosystems steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantitec reverse transcription kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Mbs263555, by MyBioSource (1 mentions)
Optima xpn 90 ultracentrifuge, by Beckman Coulter (1 mentions)
Type 70 ti rotor, by Beckman Coulter (1 mentions)
Polycarbonate ultracentrifuge bottles, by Beckman Coulter (1 mentions)
Ultracentrifuge, by Beckman Coulter (1 mentions)
Exosome depleted fbs, by Thermo Fisher Scientific (1 mentions)
Pierce bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Anti actinin 4 antibody, by Abcam (1 mentions)
Anti alix antibody, by Abcam (1 mentions)
0.2 μm filter, by Sarstedt (1 mentions)
Pkh 67 labeling kit, by Merck Group (1 mentions)
16 protocols using type 90 ti rotor
1
Viral Genomic DNA Extraction Protocol
2
Bacteriophage DNA Extraction from M. oxyfera Bioreactor
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The bacteriophage population used for sequencing was obtained from the same M. oxyfera bioreactor enrichment culture. See Gambelli et al. (2016) (link) for a full description. Bioreactor material was collected over a period of about three months, stored at 4 °C and viral particles were obtained as described before (Gambelli et al., 2016 (link)). Briefly, the aggregated microbial biomass was disrupted to free the bacteriophages and viral particles were precipitated using PEG8000 (Guo et al., 2012 (link)). Free bacteriophages present in the bioreactor supernatant medium and not within the bacterial aggregates were precipitated by iron chloride flocculation (Cunningham et al., 2015 (link)).
The two samples obtained by iron chloride flocculation and PEG 8000 precipitation were pooled together and bacteriophages were concentrated by ultracentrifugation (Optima XE90; Beckman-Coulter, High Wycombe, UK; Rotor: Type 90 Ti; Beckman-Coulter, High Wycombe, UK) at 77,000× g at 4 °C for 1 h. The pellet was resuspended in 1 ml of supernatant and the total DNA was extracted according to the protocol published by Thurber et al. (2009) (link). Using the Qubit dsDNA HS assay kit (Thermo Scientific, Waltham, MA, USA), the extracted DNA was quantified at 0.2 ng DNA.
The two samples obtained by iron chloride flocculation and PEG 8000 precipitation were pooled together and bacteriophages were concentrated by ultracentrifugation (Optima XE90; Beckman-Coulter, High Wycombe, UK; Rotor: Type 90 Ti; Beckman-Coulter, High Wycombe, UK) at 77,000× g at 4 °C for 1 h. The pellet was resuspended in 1 ml of supernatant and the total DNA was extracted according to the protocol published by Thurber et al. (2009) (link). Using the Qubit dsDNA HS assay kit (Thermo Scientific, Waltham, MA, USA), the extracted DNA was quantified at 0.2 ng DNA.
3
Isolation of NP Patient Exosomes from NLF
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Exosomes were isolated from NLF of NP patients (NPs exosomes) and healthy volunteers (normal exosomes) using a previously described method (28 (link),29 (link)) with the modifications that included differential centrifugation of NLF (6,000 × g for 30 min at 4°C and 10,000 × g for 60 min at 4°C) followed by ultrafiltration (0.2-µm filter; Sarstedt, Nümbrecht-Rommelsdorf, Germany) and qEV size-exclusion columns (iZON Science, Christchurch, New Zealand). The supernatant of NLF was then ultracentrifuged (100,000 × g) for 60 min at 4°C (Type 90 Ti Rotor; Beckman Coulter, Inc., Brea, CA, USA) to pellet the exosomes. The exosome pellets were then washed once with PBS.
4
Exosome Isolation from Conditioned Media
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Exosomes were isolated from CM as previously described18 (link). The isolation method included a penultimate centrifugation step to remove small cell debris and then ultracentrifugation at 100,000 × g for 1 h to generate an exosome pellet (Type 90 Ti rotor; Beckman Coulter, Fullerton, CA). The pellets were then washed once with phosphate-buffered saline (PBS).
5
Isolation and Characterization of Murine Macrophages and Neutrophils
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Thioglycollate-elicited peritoneal macrophages were isolated as described above. Macrophages were washed with DMEM supplemented with 10% FBS, 2 mM l -glutamine, 50 µg/mL penicillin and 50 U/mL streptomycin. Neutrophils were elicited and isolated as described above. Isolated neutrophils were centrifuged at 500× g, resuspended in PBS containing 90% Percoll and purified by ultracentrifugation using a Type 90 Ti Rotor (Beckman Coulter, Mississauga, ON, Canada) as described by others [61 (link)]. Total RNA from macrophages and neutrophils were extracted using the RNeasy mini kit (Qiagen, Toronto, ON, Canada) and RNA integrity and concentration were evaluated by measuring the absorbance at 260/280 in a spectrophotometer. In addition, 1 µg of RNA was used to prepare cDNA using the Quantitec reverse transcription kit (Qiagen, Toronto, ON, Canada). Quantitative real-time-(RT-)PCR was performed on prepared cDNA using a mixture of SYBR Green qPCR SuperMix-UDG with ROX (Thermo Fisher Scientific, Ottawa, ON, Canada) and primers listed in Table 1 . Quantitative real-time-(RT-)PCR was run on an Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Ottawa, ON, Canada).
6
Hsp90ab1 Impacts Chondrocyte Extracellular Matrix
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For in vitro experiments, cells were treated with agents for 1 day. The medium was then exchanged to remove agents, and the cells were incubated for 1 additional day with the consistent basial medium. CM was subjected to low-speed centrifugation at 2000 rpm for 10 min. The cell-free supernatants were centrifuged at 4000 rpm for 10 min and subjected to filtration with a 0.22-μm polyethersulfone membrane (Sigma, St. Louis, MO, USA). The supernatants were further centrifuged at 10,000× g for 30 min at 4 °C to remove remaining cell debris and at 100,000× g (Type 90 Ti Rotor, Beckman, Brea, CA, USA) overnight at 4 °C to remove exosomes. For in vivo experiments, CM was harvested from the fetal bovine serum-free medium and treated by a filter with a cutoff molecular weight of 3 kDa. The levels of aggrecan and type II collagen in C28/I2 chondrocytes in response to 1 µg/mL of Hsp90ab1 were determined using ELISA kits (MBS262707 and MBS263555; MyBioSource).
7
Exosome Isolation from NLF and pHNEC
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NLF was collected from NP and DNS patients, respectively, and detailed methods can be found in our previous work [11 (link)]. Exosomes were isolated by differential ultracentrifugation as we have described before [11 (link)]. Briefly, the supernatants from both NLF and pHNEC culture medium were centrifuged at 6,000 × g for 30 min and then 10,000 × g for 60min at 4°C, followed by ultrafiltration (0.2μm filter; Sarstedt, Nümbrecht-Rommelsdorf, Germany) and qEV size-exclusion columns (Izon Science, Christchurch, New Zealand). Thereafter, the supernatant was then ultracentrifuged at 100,000 × g for 60min at 4°C (Type 90 Ti Rotor; Beckman Coulter, Inc., Brea, CA, USA) to pellet the exosomes. The exosome pellets were then washed using PBS for cell experiments.
8
Virus Concentration from Wastewater
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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.
9
Exosome Isolation from Conditioned Media
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Exosomes were isolated from CM and plasma as previously described.3 , 23 The isolation method included a penultimate centrifugation step to remove small cell debris and then ultracentrifugation at 100 000 g for 1 h to generate an exosome pellet (Type 90 Ti rotor; Beckman Coulter, Fullerton, CA). The pellets were then washed once with PBS.
10
Isolation of Exosomes from Cells and Plasma
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Exosomes were separated from cells using differential centrifugation. Cells were cultured for 48 h in DMEM supplemented with 5% exosome-depleted fetal bovine serum. The subsequent cell culture supernatant was centrifuged at 500 g for 10 min to pellet cells, followed by a spin at 2,000 g for 20 min to exclude cell debris and nonviable cells.
For in vivo extraction, 1 × 106 of 231 parental, 231 LuT1, 231 LuT2, or 231 LuT3 cells were introduced into the No. 4 mammary fat pad of female NCG-HLA-A2.1 mice aged between 6 and 8 weeks. Post four weeks, blood was collected from these tumor-harboring mice after inducing deep sedation using 150 mg/kg pentobarbital. Plasma-derived exosomes were isolated with a centrifugation protocol: initial spins at 2,000 g for 20 min and 10,000 g for 20 min at 4 °C were used to remove cells and larger vesicles.
Finally, exosomes were collected by ultracentrifuging this supernatant at 110,000 g for 70 min at 4 °C using a Beckman ultracentrifuge (Type 90 Ti rotor), and the pellet was washed with PBS before being ultracentrifuged again at 11,000 g for 70 min at 4 °C.
For in vivo extraction, 1 × 106 of 231 parental, 231 LuT1, 231 LuT2, or 231 LuT3 cells were introduced into the No. 4 mammary fat pad of female NCG-HLA-A2.1 mice aged between 6 and 8 weeks. Post four weeks, blood was collected from these tumor-harboring mice after inducing deep sedation using 150 mg/kg pentobarbital. Plasma-derived exosomes were isolated with a centrifugation protocol: initial spins at 2,000 g for 20 min and 10,000 g for 20 min at 4 °C were used to remove cells and larger vesicles.
Finally, exosomes were collected by ultracentrifuging this supernatant at 110,000 g for 70 min at 4 °C using a Beckman ultracentrifuge (Type 90 Ti rotor), and the pellet was washed with PBS before being ultracentrifuged again at 11,000 g for 70 min at 4 °C.
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