The EVs were isolated using a previously described centrifugation and filtration protocol [20 (link)], with slight modifications. Briefly, for pelleting the bacteria, the broth culture was centrifuged at 5000 × g at room temperature for 10 minutes (Centrifuge 5430 R, Eppendorf AG, Germany). For removing any remnants of intact bacterial cells, the supernatant was filtered through a 0.22 μm sterile syringe filter (Millipore, Germany). The filtrate was then re-centrifuged at 125000 × g at 4° C for 3 hours (Optima™ L-XP ultracentrifuge, Beckman, USA). The obtained pellet was suspended in 300 μl sterile phosphate-buffered saline (PBS). The EVs samples were stored at -20° C until used.
Optima l xp ultracentrifuge
Manufactured by Beckman Coulter
Sourced in United States
The Optima L-XP ultracentrifuge is a laboratory equipment designed for high-speed centrifugation. It is capable of separating and isolating small particles, molecules, and cellular components from complex mixtures. The Optima L-XP ultracentrifuge operates at high speeds, up to 100,000 revolutions per minute, enabling efficient separation and purification of samples.
Automatically generated - may contain errors
Lab products found in correlation
Sterile syringe filter, by Merck Group (1 mentions)
Centrifuge 5430 r, by Eppendorf (1 mentions)
0.45 mm filter, by Merck Group (1 mentions)
Allegra x 15r, by Beckman Coulter (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Type 70 ti rotor, by Beckman Coulter (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
4 protocols using optima l xp ultracentrifuge
1
Isolation of Extracellular Vesicles from Bacterial Culture
2
Isolation and Purification of Exosomes
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Cell culture media (240 mL) were harvested from three groups (0, 1, and 2 mM) and centrifuged using a Beckman Coulter Allegra X-15R centrifuge (Beckman Coulter, Brea, CA) at 300×g (4°C) for 15 min, to remove detached cells. Supernatant was collected and centrifuged at 2000×g (4°C) for 15 min, to remove dead cells. Once again, supernatant was collected and centrifuged at 10000×g (4°C) for 30 min, to remove cellular debris. Then supernatant was collected and filtered through 0.45-mm filters (Merck Millipore, Billerica, MA) to remove apoptotic bodies and microvesicles. Clarified cell culture media were then centrifuged in a Beckman Coulter Optima L-XP Ultracentrifuge (Beckman Coulter, Brea, CA) at 110,000×g (4°C) for 90 min; swinging-bucket rotors were used to pellet exosomes. Supernatant was carefully removed, and crude exosome pellets were resuspended in 1.5 mL cold PBS (4°C). Crude exosome solution from each group was pooled in a single tube. Cold PBS was added to fill the tube completely prior to the second round of ultracentrifugation. Supernatant was carefully removed, and 100 μl cold PBS was added to each tube. Purified exosomes were gently resuspended, then stored at -80°C [19 (link), 21 (link), 22 ].
3
Analyzing Soil DNA Density by Ultracentrifugation
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To reveal the effect of 13C-straw incubation on soil DNA density, we performed density-gradient ultracentrifugation58 (link). Briefly, we centrifuged 5.1 ml of a solution composed of 3.6 µg of soil DNA (the minimum total DNA amount in all samples), 1.90 g ml−1 cesium chloride (Cat. No. 02150589-CF, MP Biomedicals, Santa Ana, CA, USA), and a gradient buffer (1 mM EDTA, 0.1 M KCl, and 0.1 M Tris-HCl), reaching a final density of 1.725 g ml−1. The solution was sealed in a polyallomer centrifuge tube (Cat. No. 342412, Beckman Coulter, Brea, CA, USA) with a cordless tube topper and centrifuged on a Vti 65.2 rotor of an Optima L-XP ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 177,000 g and 20 °C for 48 h. The solution from each centrifuged tube was then divided into twenty-four fractions (14 drops per fraction/~0.21 ml per fraction). The buoyant density of each fraction was determined by an AR200 digital refractometer (Reichert Inc., Depew, NY, USA). DNA in each fraction was then precipitated with 20 µg of glycogen and two volumes of PEG solution (30% PEG 6000 and 1.6 M NaCl), washed with 70% ethanol, and resuspended in 35 µl of ultrapure water.
4
SARS-CoV-2 M-protein Induced EV Collection
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MDA-MB-231 (ATCC HTB-26) cells (a triple-negative BCC line) and MCF-7 cells (a Luminal A BCC line [ATCC HTB-22]) were cultured in a culture dish containing Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, Waltham, MA, USA) with 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 5% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA). The cell medium was changed every two days and the cells were kept in a humidified incubator at 37°C under 5% CO2. The cells were subcultured to obtain 3.8×104 cells/ml of medium per dish via trypsinization upon reaching 80% confluence.
MDA-MB-231 cells (5×105 cells/ml) were treated with 60 pmol/ml SARS-CoV-2 M-protein (Miltenyi Biotec, Cologne, Germany) in EV-depleted FBS-containing medium for collecting EV in 5 days to collect CM and isolate EV. EV-depleted FBS-containing medium for collecting EV was prepared by ultracentrifugation using an Optima L-XP ultracentrifuge (Beckman Coulter, Inc. Brea, CA, United States), as previously described (82 (link)). Briefly, 40 ml of IMDM containing 5% FBS and 1% penicillin/streptomycin was ultracentrifuged at 140,000×g for 18 h at 4°C using a Beckman Coulter Type 70 Ti Rotor. Then, 30 ml of supernatant was collected and used as the culture medium to collect EV.
MDA-MB-231 cells (5×105 cells/ml) were treated with 60 pmol/ml SARS-CoV-2 M-protein (Miltenyi Biotec, Cologne, Germany) in EV-depleted FBS-containing medium for collecting EV in 5 days to collect CM and isolate EV. EV-depleted FBS-containing medium for collecting EV was prepared by ultracentrifugation using an Optima L-XP ultracentrifuge (Beckman Coulter, Inc. Brea, CA, United States), as previously described (82 (link)). Briefly, 40 ml of IMDM containing 5% FBS and 1% penicillin/streptomycin was ultracentrifuged at 140,000×g for 18 h at 4°C using a Beckman Coulter Type 70 Ti Rotor. Then, 30 ml of supernatant was collected and used as the culture medium to collect EV.
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