As an external validation of the proteomic results, putative exosomes were isolated from conditioned media by differential centrifugation as follows: 300 g for 10 minutes to remove cells, 2,600 g for 10 minutes to remove residual cells and debris, 10,000 g for 60 minutes to remove microparticles, and 100,000 g for 2 hours to collect exosomes in pellets. Centrifugation steps were performed using a Beckman Coulter X-14R centrifuge and a Beckman Coulter XL90 ultracentrifuge with proper rotors, open-top (Cat#: 355631) or capped (Cat#: 355618, Cat#: 355655) thickwall polycarbonate tubes (Beckman Coulter). Once isolated, the putative exosomes were resuspended in 0.2–1 ml DPBS. A solution containing the secretome isolate was dipped over clean silicon chips and liquid was evaporated after 15–60 minutes at room temperature. Silicon chips were mounted on SEM stage by carbon paste. To make surface conductive, a coating of 2–5nm gold-palladium was applied by sputtering (SPI-Module Sputtering, Argon as gas for plasma) before imaging by scanning electron microscopy (SEM Hitachi S-4700). SEM was done under low beam energies (5.0 kV). Analysis of exosome sizes were done using the SEM images via ImageJ and the density distribution of exosome diameters were obtained using R/Bioconductor Version 2.14.1.
Thickwall polycarbonate tubes
Manufactured by Beckman Coulter
Thickwall polycarbonate tubes are designed to provide a durable and reliable storage solution for various laboratory applications. These tubes are constructed from high-quality polycarbonate material, offering enhanced strength and resistance compared to standard plastic tubes. The thicker wall design enhances the overall structural integrity of the tubes, making them suitable for applications that require increased protection or stability for the contents.
Automatically generated - may contain errors
Lab products found in correlation
S 4700, by Hitachi (1 mentions)
Sigma 3 16pk centrifuge, by Merck Group (1 mentions)
Optima xe 100 ultracentrifuge, by Beckman Coulter (1 mentions)
70ti rotor, by Beckman Coulter (1 mentions)
Tla 100 fixed angle rotor, by Beckman Coulter (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Optima xpn 100, by Beckman Coulter (1 mentions)
Xl 90 ultracentrifuge, by Beckman Coulter (1 mentions)
X 14r centrifuge, by Beckman Coulter (1 mentions)
6 protocols using thickwall polycarbonate tubes
1
Isolation and Characterization of Exosomes
2
Urine EV Isolation by Centrifugation
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After thawing, 10 mL urine supernatant from each patient were vortexed to disperse EVs and protein aggregates. Then, centrifugation at 3000g during 15 min at 4 °C in a Sigma 3-16PK centrifuge (Sigma, Missouri, USA) was performed to pellet and discard urine sediment remains.
Ultracentrifugation steps described in this manuscript were performed in an Optima XE-100 Ultracentrifuge (Beckman Coulter, California, EUA) using the 70Ti rotor (Beckman Coulter, California, EUA) at 20,000g, 100,000g and 200,000g and thick wall polycarbonate tubes (Ref: 355631, Beckman Coulter, California, EUA.) with diameter: 25 mm, length: 89 mm and volume: 32 mL.
Ultracentrifugation steps described in this manuscript were performed in an Optima XE-100 Ultracentrifuge (Beckman Coulter, California, EUA) using the 70Ti rotor (Beckman Coulter, California, EUA) at 20,000g, 100,000g and 200,000g and thick wall polycarbonate tubes (Ref: 355631, Beckman Coulter, California, EUA.) with diameter: 25 mm, length: 89 mm and volume: 32 mL.
3
Amyloid-beta Aggregation Kinetics
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Aβ42 monomers (0.25 μg/μl assay buffer A) were placed on ice, or incubated for 30 min or 60 min (2 x 100 μL/condition) at 1000 rpm/37°C to form multimers. The multimer samples were transferred to thick-wall polycarbonate tubes (Beckman Coulter) and subjected to centrifugation at 20,000 rpm (15,456 × g) for 30 min (Optima Max-TL Tabletop Ultracentrifuge (Beckman Coulter); TLA-100 fixed-angle rotor (Beckman Coulter)). Supernatant (100 μL) was removed carefully from the 30 min sample so as to not disturb the pellet, and placed on ice. For the 60 min sample, supernatant was removed by pipet and the pellet was resuspended in 200μL assay buffer A. Following a second spin (20,000 rpm × 30 min), all supernatant was removed carefully, and the pellet was resuspended in 40 μL assay buffer A. The protein concentration of each sample was determined by the bicinchoninic acid (BCA) method according to the manufacturer’s directions (Pierce).
4
Subcellular Fractionation of Immune Cells
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Subcellular fractionation was performed as previously described (Segura et al., 2009 (link)). In brief, purified B cells (4–5 x 107 cells, 95–98% purity) and cDCs (4–5 x 107 cells, 90–95% purity) from spleens of WT, Marchf1-/- and Marchf8-/- mice were incubated with FITC-conjugated anti-CD19 and anti-B220 mAb (B cells) or anti-CD11c, anti-CD45.2, anti-CD49d and anti-MHC I mAb (cDCs). mAb-labelled cells were homogenized in the presence of cOmplete™ protease inhibitors (Roche) by mechanical disruption using a cell-cracker (HGM Laboratory equipment). Homogenized preparations were centrifuged at low speed to obtain post-nuclear supernatant (PNS). Surface-labelled plasma membrane (PM) microsomes were isolated by magnetic immunoaffinity using anti-FITC mAb-coated magnetic beads (Miltenyi Biotec) and concentrated by ultracentrifugation in thickwall polycarbonate tubes (Beckman Coulter). PNS, with the PM fraction removed, was likewise ultracentrifuged to sediment the “intracellular compartments” (IC) fraction.
5
Ultracentrifugation for Extracellular Vesicle Isolation
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Clarified plasma was transferred to 4 mL (38 mL for larger plasma volumes) Thickwall Polycarbonate Tubes (Beckman Coulter®), and filtered PBS was added to a final volume of 3.8 mL (16 mL for larger plasma volumes). Next, the samples were centrifuged in a Beckman Coulter Optima™ XPN-100 ultracentrifuge at 100,000 × gavg for 70 min at 4°C using a Type 70 Ti Fixed-Angle rotor (k-factor = 186.1) to pellet sEVs. The pellet was resuspended to the initial volume in filtered PBS as a wash and centrifuged a second time at 100,000 × gavg for 70 min at 4°C. The final pellet was resuspended in filtered PBS and subjected directly to downstream analyses.
6
Exosome Isolation by Differential Centrifugation
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