Plasma and extrapallial fluid samples were centrifuged at 300,000 g, 4°C, for 120 min using a Beckman L8-70M Ultracentrifuge with a Type 70 Ti Fixed Angel Rotor. The protein pellets were dissolved in 1.39 g/ml CsCl solution in 0.02 M Tris-HCl, pH 8.0. The solution was centrifuged at 5,000 g, 4°C for 15 min to remove insoluble particles and the supernatant was then centrifuged at 200,000 g, 4°C for 20 h using a Beckman L8-70M Ultracentrifuge with a SW 55 Ti Swinging-Bucket rotor. After centrifugation, purified protein particles were collected from the turbid layer in the centrifuge tube using a 1 ml syringe equipped with a 25-gauge needle. The collected samples were stored at -20°C before analyses for morphology by transmission electron microscopy and for protein composition by polyacrylamide gel electrophoresis. The purified protein particles were observed after negative stain with 0.5% uranyl acetate at the Louisiana State University Shared Instrumentation Facility in Baton Rouge, Louisiana using a JEOL-1400 Transmission Electron Microscope (JEOL USA, Inc., Peabody, MA, United States).
L8 70m ultracentrifuge
Manufactured by Beckman Coulter
Sourced in United States
The L8-70M Ultracentrifuge is a high-speed centrifuge designed for a variety of laboratory applications. It can achieve rotational speeds up to 70,000 rpm, enabling the separation and analysis of small particles and macromolecules. The L8-70M provides precise temperature control and a range of rotor options to accommodate different sample volumes and densities.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (2 mentions)
Sw41ti rotor, by Beckman Coulter (2 mentions)
Type 45 ti rotor, by Beckman Coulter (1 mentions)
150 mm cell culture dishes, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Sw28 rotor, by Beckman Coulter (1 mentions)
Small animal optical imaging system in vivo xtreme 2, by Bruker (1 mentions)
Nanosight ns300, by Malvern Panalytical (1 mentions)
1400 transmission electron microscope, by JEOL (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Fgfr1, by Horizon Discovery (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Cyclohexamide, by Merck Group (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr assay, by Bio-Rad (1 mentions)
Myiq2 real time pcr detection system, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rotavapor r 114, by Büchi (1 mentions)
18 protocols using l8 70m ultracentrifuge
1
Purification of Protein Particles
2
Polysome Profile Analysis of HuH-7 Cells
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HuH-7 sgRNA-ctrl and sgRNA-24 cells were incubated with 100 µg/ml cycloheximide (Sigma) in the growth media for 5 min at 37°C and 5% CO2. Cells were washed once in ice-cold PBS containing 100 µg/ml cycloheximide. Cells were then scraped in 5 ml of ice-cold PBS containing 100 µg/ml cycloheximide and pelleted. Cell pellets were lysed in buffer containing 10 mM Tris-HCl (pH 8), 150 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 20 mM DTT, 150 µg/ml cycloheximide, and 640 U/ml Rnasin for 30 min on ice. Lysates were centrifuged at 10,000 x g for 5 min at 4°C. The supernatant (~300 ul) were adjusted by OD260 (to OD260 of ~15) and loaded onto a 10–50% sucrose gradient before centrifugation at 37,500 rpm for 2.5 hrs at 4°C in a Beckman L8-70M ultracentrifuge. Samples were separated on an ISCO gradient fractionation system to evaluate polysome profiles.
3
Exosome Isolation from RAW264.7 Cells
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Conditioned medium of RAW264.7 cultured in fetal bovine serum (FBS)–free Dulbecco's Modified Eagle Medium (DMEM) were collected for 48 h and exosomes were purified by several centrifugation and filtration steps, as described previously (15 (link)). Briefly, the supernatant was centrifuged at 2,000 × g for 10 min and 16,000 × g for 30 min, followed by filtration through a 0.22-μm filter to eliminate cells, dead cells, and cellular debris. For exosome purification, the supernatant was ultracentrifuged at 160,000 × g for 180 min (L8-70M ultracentrifuge; Beckman Coulter, Brea, California). The exosome pellets were re-suspended in PBS and stored at −80˚C.
4
Isolation of Small Extracellular Vesicles from Prostate Cancer Cells
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As published previously,62 (link) for sEV isolation, PrCa cells (PC3 and DU145) were plated in 150 mm cell culture dishes (ThermoScientific, 130183) in their respective complete medium, as described above. After 48 hours of incubation at 37°C, cells were washed with PBS and incubated with serum-free medium (complete media devoid of FBS) for the next 48 hours. The sEVs were isolated by high-speed differential ultracentrifugation of the supernatant (SN) collected after 48 hours of serum-starvation. The dead cells and cell debris were spun down from SN at 2000 xg, 4°C for 20 minutes. The SN collected was spun at 10,000 xg, 4°C for 35 minutes. Next, the SN collected without disturbing the 10,000 xg pellet was spun at 100,000 xg, 4°C for 70 minutes; the pellet was washed in 40 mL PBS followed by a second spin at 100,000 xg, 4°C for 70 minutes. The 10,000 xg and 100,000 xg centrifugation were done in a Beckman Type 45Ti rotor using a Beckman L8-70M Ultracentrifuge. The final sEV pellets were resuspended in 100 μL PBS.
5
Lentiviral Particle Production and Titration
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Lentiviral particles were produced using a four plasmid tat-independent packaging system delivered into cells by calcium phosphate transfection [27 (link)]. At approximately 16 h post-transfection, the medium was replaced with fresh DMEM supplemented with 5 mM sodium butyrate. The media was collected after 24 h, and the virus-containing media was filtered through a 0.45-μM filter (MillexHV Millipore) to remove cell debris. Viral concentration was achieved by centrifugation at 20,000g for 2 h at 4 °C in a Beckman L8-70M Ultracentrifuge using an SW28 rotor (Beckman). Following centrifugation, the supernatant was removed, and the viral pellets were resuspended in 1/100th of the original volume in DMEM/10% FCS. Viral titres were determined by testing transduction levels on HeLa cells using serially diluted virus. Cells were collected 48 h post-transduction and analysis by flow cytometry using an LSR Fortessa (BD). Percentages of GFP-positive cells at each virus dilution were evaluated using FlowJo version 9.4 (Treestar).
6
Exosome Isolation and Characterization
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Serum was 5× diluted with phosphate‐buffered saline (PBS) and cell debris, and organelles were eliminated by centrifugation at 1000g for 10 min, 4 °C. The supernatant fraction was further centrifuged at 16 000g for 30 min. The second supernatant was sterile filtered through a 0.22‐μm filter. Exosomes were pelleted at 120 000g for 90 min at 4 °C (L8‐70 M ultracentrifuge, Beckman‐Coulter, Indianapolis IN). Finally, the exosome pellets were re‐suspended in 100 to 400‐μL PBS. Protein content was quantitated by using a Bradford protein assay. Exosome concentration and size (Figure S3 ) were measured with NanoSight instruments, which performs nanoparticle tracking analysis (NanoSight NS300, Malvern Instruments, Inc. Westborough, MA). In vivo exosome distribution imaged was taken with the Bruker Small Animal Optical Imaging System (In‐Vivo Xtreme II; Billerica, MA).
7
Lentiviral Transduction of Cell Lines
8
Polysome Profiling and qPCR Analysis
9
Lentiviral Particle Production and Concentration
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Three helper plasmids (i.e., pRSV-REV, pMDLg/pRRE, and VSV-G; 7–8 µg each) and corresponding expression vectors (15–20 µg) were co-transfected with polyethylenimine (PEI) into 70–80% confluent HEK 293T (containing SV40 T-antigen to facilitate virus production) cells plated on 10 cm dishes. At 8–10 h post-transfection, the culture medium was exchanged completely, and the supernatant containing lentivirus particles was collected after 36 h and 60 h. The supernatant was then pooled and spun at ~800 × g for 6–8 min to remove any HEK cell debris. The supernatant was then spun at ~120,000 × g for 2 h at 4 °C (Beckman L8–70M ultracentrifuge equipped with SW41Ti rotor). The viral pellets were resuspended in ~50-100 µl DMEM media, stored overnight at 4 °C, subsequently aliquoted and frozen at -80 °C prior to experimental use.
10
pH-Responsive Liposome Preparation
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The thin film method used to prepare pH-responsive liposomes has been described previously [22 (link)]. In brief, the DOPE, CHEMS, and DSPE-PEG750 or DSPE-PEG2000 (molar ratio of 6:4 and 6:4:0.1, respectively) were dissolved in a 50:50 methanol:chloroform (v/v) solvent mixture in a round bottom flask. The solvents were removed under reduced pressure on a Büchi Rotavapor R-114 with vacuum pumpV-500 (Büchi Labortechnik, Flawil, Switzerland) at 45 °C and 60 rpm to obtain a thin lipid film that was left to completely dry at 50 mbar for 1 h. The dry lipid film was hydrated with a buffered solution of 80 mM calcein for the calcein containing liposomes or PBS (pH 7.4) for the non-calcein containing liposomes. The liposomes were stepwise hand extruded through Nucleopore® polycarbonate membranes (0.8, 0.4, 0.2, and 0.1 µm pore sizes, three cycles for each size).
For calcein containing liposomes, the extravesicular calcein was removed by ultracentrifugation at 300,000× g for 1 h at 10 °C (L8-70M Ultracentrifuge; Beckman Instruments, Palo Alto, CA, USA). The supernatant was carefully removed before the pellet was gently resuspended in PBS using a bench vortex mixer and centrifuged for an additional 1 h. The supernatant was then removed and the pellet gently resuspended in PBS by vortexing to obtain the final liposomal formulations.
For calcein containing liposomes, the extravesicular calcein was removed by ultracentrifugation at 300,000× g for 1 h at 10 °C (L8-70M Ultracentrifuge; Beckman Instruments, Palo Alto, CA, USA). The supernatant was carefully removed before the pellet was gently resuspended in PBS using a bench vortex mixer and centrifuged for an additional 1 h. The supernatant was then removed and the pellet gently resuspended in PBS by vortexing to obtain the final liposomal formulations.
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