The 15 mL of conditioned media from the cell chamber of the bioreactor was centrifuged at 2,000 xg for 10 min to remove cells and other debris. The supernatant was then centrifuged at 10,000 xg for 30 min (JA.30-50 Ti rotor, Avanti, Beckman Coulter) to pellet the large EVs (also known as microvesicles) and this pellet was resuspended in 500 µL PBS and stored. The supernatant was then ultracentrifuged at 100,000 xg for 70 min (JA.30-50 Ti rotor, Avanti, Beckman Coulter) to yield a crude small EV pellet. This pellet was resuspended in 700 µL PBS and stored at -80 0 C until needed, minimising the number of freeze thaws. 500 µL of crude small EVs were loaded onto a 35 nm qEV Original SEC column (Izon Science Ltd.), and fractions 7 through 23 were collected using an automated fraction collector (Izon Science Ltd, 500 µL per fraction). A high sensitivity BCA assay (Pierce, ThermoFisher Scientific) was performed for each collected fraction to determine their protein concentration. Once EV-rich fractions were determined, only those EV-rich fractions (F8-F10) were collected and pooled in subsequent isolations.
Ja 30.50 ti rotor
Manufactured by Beckman Coulter
Sourced in United States
The JA-30.50 Ti rotor is a high-performance centrifuge rotor designed for use with Beckman Coulter's ultracentrifuge systems. The rotor is made of titanium and is capable of reaching maximum speeds of 30,000 revolutions per minute (rpm). It is suitable for a wide range of applications that require high-speed centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Avanti j 30i centrifuge, by Beckman Coulter (3 mentions)
Polyethersulfone syringe filters, by Techno Plastic Products (1 mentions)
Sw55ti rotor, by Beckman Coulter (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Tla 100.3 rotor, by Beckman Coulter (1 mentions)
Sw28 rotor, by Beckman Coulter (1 mentions)
Avanti j 301 centrifuge, by Beckman Coulter (1 mentions)
Polycarbonate tube, by Beckman Coulter (1 mentions)
Vivaspin 500 column, by Sartorius (1 mentions)
Pes syringe filter, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Vivaflow 200, by Sartorius (1 mentions)
Qev original sec column, by Izon Science (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
9 protocols using ja 30.50 ti rotor
1
Isolation and Fractionation of EVs
2
Phage Purification and Concentration
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Phage lysate, obtained after complete lysis of the bacteria, was centrifuged at 4500× g for 30 min at 4 °C and filtered through 0.45 μm pore-sized polyethersulfone syringe filters (Techno Plastic Products, Trasadingen, Switzerland) to remove bacterial debris. Phages were pelleted by centrifugation at 54,000× g for 2.5 h at 4 °C in a JA-30.50 Ti rotor (Beckman, Brea, CA, USA). For preparation of pellets from 3 mL of phage lysates, conical tubes (part no. 358119, Beckman) and adapters (part no. 358153, Beckman) with an SW 55 Ti rotor (Beckman) were used. The resulting pellet was resuspended in 350 μL of phage buffer (5 × 10−2 mol/L Tris pH 8.0, 10−2 mol/L CaCl2, 10−2 mol/L NaCl) overnight at 4 °C. For resuspending the pellets from 3 mL samples, 30 μL of phage buffer was used.
3
Production of CuMVTT Virus-Like Particles
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The production of CuMVTT-VLP was described in detail in Zeltins et al. [28 (link)]. Briefly, Escherichia coli C2566 cells (New England Biolabs, Ipswich, MA, USA) were transformed with the CuMVTT coat protein (CP) gene-containing plasmid pET CuMVTT. The expression was induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The resulting biomass was collected by low-speed centrifugation and was frozen at −20 °C. After thawing on ice, the cells were suspended in the buffer containing 50 mM sodium citrate, 5 mM sodium borate, 5 mM EDTA, and 5 mM 2-mercaptoethanol (pH 9.0, buffer A) and were disrupted by ultrasonic treatment. Insoluble proteins and cell debris were removed by centrifugation (13,000 rpm, 30 min at 5 °C, JA-30.50 Ti rotor, Beckman, Palo Alto, CA, USA). The soluble CuMVTT CP protein in clarified lysate was pelleted using saturated ammonium sulfate (1:1, vol/vol) overnight at 4 °C. Soluble CuMVTT CP-containing protein solution was separated from the cellular proteins by ultracentrifugation in a sucrose gradient (20–60% sucrose; ultracentrifugation at 25,000 rpm for 6 h at 5 °C (SW28 rotor, Beckman)). After dialysis of CuMVTT-containing gradient fractions, VLPs were concentrated using ultracentrifuge (TLA100.3 rotor, Beckman, at 72,000 rpm for 1 h, +5 °C) or by ultrafiltration using Amicon Ultra 15 (100 kDa; Merck Millipore, Cork, Ireland).
4
Synthesis of Homopolymer Microgels
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The homopolymer microgels of NNPAM, NIPAM, and NIPMAM were synthesized via conventional precipitation polymerization without surfactant. All syntheses were performed in a 250 mL three-neck flask equipped with a reflux condenser, mechanical stirrer (210 rpm), and a nitrogen inlet. The monomer (11.05 mmol) and the cross-linker N,N’-methylenebisacrylamide (BIS) (2.5 mol%, 5.0 mol%, 6.75 mol%, 7.5 mol%, 8.75 mol%, 10.0 mol%, 11.25 mol%, 12.5 mol%, 13.75 mol%, 15.0 mol% respective to the total monomer amount) were dissolved in 150 mL purified water and heated to 70 °C under continuous stirring and purged with nitrogen. After 1 h the polymerization was initiated by the addition of 2 mL of the 0.2 M solution of APS and left to proceed for 4 h at 70 °C. Subsequently, the solution was cooled to room temperature and stirred overnight. For purification, all samples were treated by four cycles of centrifugation, decantation, and redispersion in purified water using a JA-30.50 Ti Rotor in an Avanti J-30I centrifuge (Beckman Coulter, Brea, CA, USA) at 20,000 rpm and 25 °C.
5
Placental Exosome Isolation Protocol
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Placenta extracts preparations were obtained from total placentas. Supernatants were subjected to sequential centrifugation: twice at 10,000× g for 40 min at 4 °C and once for 16,500× g for 20 min (Beckman Coulter Avanti-J-301 centrifuge, JA-30.50 Ti rotor), the supernatant was filtered through filter 0.22 microns. The filtered supernatant was ultracentrifuged at 100,000× g for 2 h. After the first centrifugation, the pellet was resuspended in 8 mL of TBS. The resuspended pellet was ultracentrifuged twice at 100,000× g for 2 h (Beckman L8-M centrifuge, SW-60 rotor (Brea, CA, USA). The precipitate was resuspended, filtered through a filter (0.1 µm) and used for additional purification. For additional purification of exosomes, gel filtration on columns with Sepharose 4B was used.
6
Extracellular Vesicle Isolation Protocol
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Bacterial cells were removed from the culture broths by centrifugation twice at 7,000 × g for 10 min at 4°C followed by filtration through 0.22 μm PES syringe filter (Merck Millipore), and then concentrated using 100 kDa Vivaflow 200 (Sartorius AG). Crude input EV was prepared from the cleared supernatants by ultracentrifugation in polycarbonate tube (29 x 104 mm; Beckman Coulter) at 75,000 × g for 2.5 hr at 4°C in Avanti J-30I centrifuge with JA-30.50 Ti rotor (Beckman Coulter). Resulting EV pellets were resuspended in 1 ml of PBS (Sigma-Aldrich), filtered through 0.22 μm filter and further concentrated using 100 kDa Vivaspin 500 columns (Sartorius AG) before storage at −80°C [21 ]. These crude input EV preparations were used for further purification by either DGC or SEC as per [17 ]. Details are shown below and Supplementary Data 1.
7
EV Isolation from Endometrial Cells
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Ultracentrifugation was assessed for its efficiency in isolating EVs from conditioned culture medium of endometrial epithelial cells. Conditioned culture medium was first centrifuged at 300 × g for 10 min at 4 °C, then at 2,000 × g for 20 min at 4 °C to remove cells. Supernatant was then transferred to polycarbonate tubes and centrifuged at 10,000 × g for 30 min at 4 °C to remove cell debris. Subsequently, supernatant was collected and centrifuged twice at 100,000 × g for 70 min at 4 °C to pellet EVs, using a Beckman-Coulter JA-30.50 Ti Rotor. Pellet was resuspended in either PBS or RIPA lysis buffer depending on the characterization method.
8
Exosome Isolation from Goat Milk
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Exosomes were isolated from commercial fresh pasteurized semi-skimmed goat milk (El Cantero de Letur, Albacete, Spain) by successive centrifugations at 4 °C in 30 mL polycarbonate centrifuge tubes, using a Ja 30,50 Ti rotor (Beckman Coulter Instruments, Brea, CA, USA). Briefly, milk was centrifuged for 10 min at 5,000× g, 35 min at 13,000× g, and 15 min at 35,000× g in order to remove contaminants such as fat globules (MFGs) and cell debris. Additionally, microbial rennet was used to precipitate milk casein. The supernatant was ultracentrifuged at 100,000× g and 4 °C for 70 min to precipitate the exosomes (50–150 nm). The resultant pellet was washed three times with phosphate-buffered saline (PBS 1X) and then purified by size exclusion chromatography (SEC) using PD-10 columns (GE Healthcare Bio-Sciences AB, Chicago, IL, USA). Pure exosomes were ultracentrifuged again at 100,000× g for 90 min, and the exosome pellet was dispersed in 100 µL of PBS 1X. The protein content of the final sample was quantified using the Coomassie–Bradford assay and stored in aliquots at −20 °C until use.
9
NIPAM Microgels Synthesis via Precipitation Polymerization
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The homopolymer microgels of NIPAM were synthesized via conventional precipitation polymerization without surfactant. All syntheses are performed in 250 mL three-neck flasks equipped with a reflux condenser, mechanical stirrer (210 rpm), and a nitrogen inlet. The monomer (11.05 mmol) and the cross-linker N,N-methylenebisacrylamide (5.0 mol%, (
10.0 mol%, 15.0 mol% with respect to the total monomer amount) are dissolved in 150 mL purified water and heated to 70 • C under continuous stirring and purged with nitrogen for 1h. Subsequently, the polymerization is initiated by the addition of 2 mL of the 0.2 M solution of APS and left to proceed for 4 h at 70 • C. Subsequently, the solution is cooled to room temperature and stirred overnight. For purification, all samples are treated by four cycles of centrifugation, decantation, and redispersion in purified water using a JA-30.50 Ti Rotor in an Avanti J-30I centrifuge (Beckman Coulter, Brea, CA, USA) at 20,000 rpm and 25 • C.
10.0 mol%, 15.0 mol% with respect to the total monomer amount) are dissolved in 150 mL purified water and heated to 70 • C under continuous stirring and purged with nitrogen for 1h. Subsequently, the polymerization is initiated by the addition of 2 mL of the 0.2 M solution of APS and left to proceed for 4 h at 70 • C. Subsequently, the solution is cooled to room temperature and stirred overnight. For purification, all samples are treated by four cycles of centrifugation, decantation, and redispersion in purified water using a JA-30.50 Ti Rotor in an Avanti J-30I centrifuge (Beckman Coulter, Brea, CA, USA) at 20,000 rpm and 25 • C.
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