Exosomes were isolated from the fresh or frozen cell culture supernatants as previously described 30 . Briefly, cells were removed by centrifugations at 300g for 10 min (TX-400 rotor, ST16R, Thermo Fisher, Massachusetts, USA). Subsequently, supernatants were centrifuged at 2,000g for 10 min to remove smaller cellular debris and at 10,000g for 30 min to remove apoptotic bodies (F15-6x100y rotor, ST16R, Thermo Fisher, Massachusetts, USA). Exosomes were then harvested by ultracentrifugation at 100,000g for 70 min in a swing rotor (SW32Ti, XPN-100, Beckman, California, USA). All centrifugation steps were performed at 4℃, and isolated exosomes were suspended in phosphate-buffered saline (PBS) and stored at -80℃. The protein content of exosome suspension was quantified using the bicinchoninic acid (BCA) assay (Beyotime, P0010, Beijing, China). The representative markers CD63 (1:1000, Abcam, California, USA) and CD81 (1:1000, Abcam, California, USA) of exosomes were assayed by Western blotting. Furthermore, the morphology and size distribution of exosomes were detected by transmission electron microscope (TEM) (JEM-1400, JEOL Ltd., Japan) and nanoparticle tracking analysis (Nanosight LM10, Malvern, Worchestershire, UK), respectively. The protein content of the MSC-Exo preparations was normalized to total protein content as quantified by BCA assay.
Xpn 100
Manufactured by Beckman Coulter
Sourced in United States
The XPN-100 is a high-performance centrifuge designed for laboratory applications. It features a compact design and can accommodate a range of sample volumes. The XPN-100 is capable of reaching high speeds to facilitate efficient sample separation and processing.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer, by Zeiss (2 mentions)
0.22 μm membrane filter, by Merck Group (2 mentions)
F15 6x 100y rotor, by Thermo Fisher Scientific (1 mentions)
Tx 400 rotor, by Thermo Fisher Scientific (1 mentions)
St16r, by Thermo Fisher Scientific (1 mentions)
Jem 1400, by JEOL (1 mentions)
P0010, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Beckman Coulter (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Mini26 1kt, by Merck Group (1 mentions)
Rpmi 1640, by Sartorius (1 mentions)
Sorvall st 16r, by Thermo Fisher Scientific (1 mentions)
0.22 μm pore size filter, by Merck Group (1 mentions)
Vacuum filter system, by Corning (1 mentions)
Total exosome rna and protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Avanti j e, by Beckman Coulter (1 mentions)
Pkh67 membrane dye, by Merck Group (1 mentions)
H 7500, by Hitachi (1 mentions)
Nh4 2so4, by Merck Group (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
20 protocols using xpn 100
1
Isolation and Characterization of Exosomes
2
Isolation of Umbilical Cord MSC-Derived Exosomes
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Human umbilical cord tissue was obtained from 5 healthy consenting mothers (age 25-30) using standard procedures performed according to the Guideline of the Ethical Committee of the Xi'an Central Hospital and in keeping with National Institutes of Health Guidelines. MSCs were obtained from enzymatic digestion of Wharton's Jelly tissue and expanded using complete culture medium (CCM) consisting of α-minimum essential medium (α-MEM; Gibco, New York, USA) and 16.5% fetal bovine serum (FBS) (Gibco, New York, USA). The 2nd or 3rd passage of MSCs were assessed for meeting the minimum criteria established by the Mesenchymal and Tissue Stem Cell Committee of International Society for Cellular Therapy, as described in our previous report 28 (link). For the culture conditions preparing for exosome isolation, exosome-depleted fetal bovine serum (FBS) was prepared by 18h ultracentrifugation of regular FBS at 100,000g using an SW32Ti rotor (XPN-100, Beckman, California, USA) 29 (link). The top layers of the FBS supernatant (approx. 9/10) were retained and used in the subsequent culture. When the 5th passage of MSCs reached about 50-70% confluency, the medium was replaced with CCM containing exosome depleted FBS. After 24-48 h, the supernatants were harvested and processed immediately to isolate exosomes or frozen at -80°C for storage.
3
Isolation and Characterization of Extracellular Vesicles from Bone Marrow Stromal Cells
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Logarithmically growing BMSCs were seeded into stem cell culture medium with 10% EV-free fetal bovine serum (FBS) (SA102.02, CellMax Life, San Diego, Califonia) and incubated in a cell incubator at 37 °C and 5% CO2. After 24 h, the supernatant of BMSCs was collected and centrifuged (300 × g, for 10 min; 2000 × g for 20 min) to remove the cells and cell fragments. Following three times of ultracentrifugation (XPN100, Beckman Coulter, Brea, CA) (100,000 × g for 90 min, for 90 min, and for 120 min), EVs were obtained, re-suspended in 100 μL sterile PBS and stored at −80 °C for later use. Information about EVs in this study has been submitted to EV track (https://www.evtrack.org ) with the reference number of EV220156.
4
Extracellular Vesicle Isolation by Iodixanol Gradient
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Iodixanol density gradient medium (StemCell) was prepared in ice‐cold PBS immediately before use to generate a discontinuous step gradient (12–28%). The 28% iodixanol solution was added to the bottom of a centrifugation tube, and of iodixanol at decreasing concentrations in PBS were carefully layered on top of the iodixanol solution, yielding the complete gradient. Identical gradients without sample were generated and ultracentrifugated by the same way for later determination of fraction densities. The density of fractions was measured by refractometry. Crude EV pellets resuspended in ice‐cold PBS were added to the top of the centrifugation tube. The gradient was subjected to ultracentrifugation at 120,000 × g for 16 h at 4°C using Beckman XPN‐100 with an SW 41 Ti swinging bucket rotor. Nine individual 1‐ml fractions were collected from the top of the gradient. For WB analysis, each individual 1 ml fraction was transferred to a new ultracentrifugation tube, diluted in PBS and subjected to ultracentrifugation at 120,000 × g for 4 h at 4°C using an SW 32 Ti swinging bucket rotor. The resulting pellets were lysed in 5 × SDS buffer on ice and boiled for 10 min at 100°C.
5
Thylakoid Membrane Purification and Analysis
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One milligram of purified thylakoid membranes was solubilized in 1% DM to a final concentration of 1 mg/ml, and the sample was loaded on a 10.5 ml 0~1.0 M SDG (consisting of 20 mM Tricine-NaOH [pH 7.8], and 0.05% DM). Two green bands were obtained after centrifugation at 288,000 × g for 6 h in a swinging bucket rotor (Beckman, XPN-100, USA) at 4 °C, and the bands were collected with syringes and denatured at 60 °C for 10 min36 (link).
6
EV Isolation and Uptake Analysis
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EVs were collected essentially as previously described (Théry et al., 2018 (link)). To isolate EVs from cell culture supernatants, the culture supernatant of CNE2 cells was subjected to differential centrifugation at 500 × g for 10 min, 3000 × g for 60 min, and 10,000 × g for 60 min at 4°C. After filtering through a 0.22 m filter, the supernatant was transferred to an ultracentrifuge tube. Next, the supernatant was centrifuged at 100,000 × g (XPN‐100, Beckman Coulter) for 90 min at 4°C. CNE2 cells were cultured in RPMI 1640 (Biological Industries Israel Beit‐Haemek, 01‐100‐1ACS) without FBS for 24 h prior to collect the culture supernatant. The isolated EVs were resuspended in phosphate‐buffered saline (PBS). To detect the uptake of EVs by recipient cells, we used a PKH‐26 labeling kit (Sigma–Aldrich, MINI26‐1KT) to label EVs and then cocultured them with HUVECs for 2 h. Finally, the HUVECs ‐nuclei were stained with Hoechst. Images were acquired using a confocal microscope (Axio Observer, Zeiss, Göttingen, Germany,). Further, to detect the uptake of HAX1 of EVs by HUVECs, we used an upper and lower chamber cocultivation system. After 48 h, the HUVECs in the lower cavity were stained with Hoechst stain and imaged.
7
Isolation and Purification of Bacterial Outer Membrane Vesicles
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Escherichia coli MG1655 in LB medium was incubated in a shaking incubator at 220 rpm at 37 °C overnight. Then, bacterial cells were removed by centrifuging at 10,000 × g for 10 min at 4 °C (Sorvall ST16R, Thermo Scientific). The obtained supernatant was filtered by 0.45 μm filters (Vacuum Filter System, Corning) and concentrated by centrifugal filters with a molecular weight cutoff (MWCO) of 100 kDa (Millipore). The concentrated supernatant was filtered again through 0.22 μm pore size filters (Millipore) to remove any remaining debris or bacteria. Then, the concentrated medium was centrifuged at 150,000 × g for 3 h at 4 °C (XPN 100, Beckman). The supernatant was removed and the pellet was resuspended in PBS and stored at -80 °C. The protein concentration of OMVs was determined by Micro BCA Protein Assay Kit.
8
Exosome Isolation and Characterization Protocol
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We obtained exosomes according to previous descriptions 31 (link)-33 (link). The cell supernatant was centrifuged by differential centrifugation at 300 × g, 3000 × g, 6000 × g, 10000 × g, and cell-free supernatant was subsequently ultracentrifuged at 100000 × g for 60 min at 4 °C (XPN-100, Beckman, USA). The products were resuspended and washed twice by PBS without exosomes and confirmed by the ultracentrifugation again. BCA protein assay kit (PIERCE Co., USA) and Total Exosome RNA and Protein Isolation Kit (Invitrogen Life technologies, USA) were used to estimate the content of protein and RNA in exosome pellets. For experiments in vitro, 2 μg exosomes were added to about 2 × 105 recipient cells. All the isolated exosomes above were stored at -80 °C.
9
Exosome Isolation from Conditioned Media
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Approximately 5 × 106 cells were seeded in cell culture dishes. To make the data more accurate, the same number of cells were seeded in different comparison groups each time. Then, cells were cultured in normal medium until 80% confluent, followed by 3 PBS washes and 18 mL of serum-free medium culturing for an additional 40 h. Next, the serum-free conditioned medium was harvested and centrifuged sequentially at 300 × g and 2000 × g for 10 min to remove cells and cell debris, respectively. Small cell debris and larger microvesicles were removed by 10,000 × g centrifugation for 30 min (Avanti J-E, Beckman, USA). Finally, the exosome-rich pellet was collected after ultracentrifugation at 167,000 × g for 70 min (XPN-100, Beckman, USA). The exosome-rich pellet was washed by PBS and ultracentrifuged at 167,000 × g for 70 min again [42 (link), 43 (link)]. Exosomes were aliquoted for the following experiments.
10
Isolation of BMSC- and BEC-Derived EVs
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EVs derived from BMSCs and BECs were isolated by ultrafiltration and Exo-Prep kit (HansaBioMed Life Sciences) according to the manufacturer’s protocol. Briefly, BMSCs and BECs were seeded into 75 cm3 flasks and cultured to 50–60% confluence. Then, EVs-deprived fetal bovine serum (EVs-free FBS) was prepared by ultracentrifuge at 100,000 g for 18 h under 4 ℃ (XPN-100, Beckman Coulter) according to a previous report [26 (link)] and then was added into culture medium when cells grew to 70–80% confluence. Eventually, cells were allowed to grow for another 24 h. After that, the supernatants were collected, centrifuged under 1000 g for 5 min, filtered by 0.22 μm membrane filters (Millipore), and then concentrated by ultrafiltration spin columns (28932358, Cytiva). Exo-prep reagent was added to the concentrated supernatant and left to stand for 1 h on ice until centrifuged at 10,000 g for another 1 h under 4 ℃. The EVs-enriched precipitation was washed with cold PBS 3 times and centrifuged again at 10,000 g under 4 ℃ for 5 min to fully abandon the supernatant and reagent. The final precipitation, including BEC-EVs or BMSC-EVs, was resuspended in 200 μl PBS and stored at − 80 ℃ refrigerator till further experiments. All samples were filtered again using 0.22 μm membrane filters to ensure sample sterility before administration.
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