α-minimal essential medium (α-MEM) containing 20% FBS was centrifuged at 200,000 ×g for 18 h to deplete Exo. MSCs from passages 4-6 were cultured using 10% Exo-removed FBS for Exo collection. 2×106 MSCs were cultured in 100 mm dishes in normoxia and hypoxia condition for 72 h and 10 mL supernatant was collected. Exo were then isolated from the harvested supernatant according to a previous study 27 . Briefly, the supernatant was centrifuged at 300 ×g for 10 min, 1200 ×g for 20 min, then 10,000 ×g for 30 min at 4 ℃ and then filtered using a 0.22 µm filter (Millipore, Billerica, MA, USA) to remove cells and debris. The filtrate was centrifuged at 140,000 ×g for 90 min at 4 ℃ using a Type Ti70 rotor using an L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA). Applying PBS to resuspend the Exo pellet and centrifuged again at 140,000 ×g for 90 min. Afterwards, the pelleted Exo were resuspended in PBS and tested using a BCA Protein Assay kit (Thermo Fishier Scientific, USA). To detect Exo markers and the negative markers, Western blotting was applied with anti-Alix and anti-TSG101 antibodies (Abcam, Cambridge, UK) 28 (link).
L 80xp ultracentrifuge
Manufactured by Beckman Coulter
Sourced in United States
The L-80XP ultracentrifuge is a high-performance laboratory equipment designed for separating and analyzing complex biological samples. It utilizes centrifugal force to separate particles, cells, and macromolecules based on their size, shape, and density. The L-80XP can achieve high rotational speeds, enabling efficient separation and purification of a wide range of materials, such as proteins, nucleic acids, and organelles. This equipment is a versatile tool for researchers and scientists working in various fields, including biochemistry, molecular biology, and cell biology.
Automatically generated - may contain errors
Lab products found in correlation
0.22 μm filter, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
0.22 m filter, by Merck Group (1 mentions)
Flotillin 1, by Proteintech (1 mentions)
Ha tag, by Proteintech (1 mentions)
Bca protein detection kit, by Thermo Fisher Scientific (1 mentions)
Anti alix, by Abcam (1 mentions)
Anti calnexin antibody, by Abcam (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Beckman Coulter (1 mentions)
F 12k medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
8 protocols using l 80xp ultracentrifuge
1
Exosome Isolation from MSC Conditioned Media
2
Isolation and Characterization of Exosomes from Mesenchymal Stem Cells
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Mesenchymal stem cells were derived from mouse bone marrow tibias and femurs and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco) without exosomes (FBS was centrifuged at 200,000g for 18 h to deplete exosomes) and then incubated at 37 °C in 5% CO2. To label exosomes with tdTomato, cells were stably transduced with packaged lentivirus vectors to express tdTomato fused with the palmitoylation sequence of growth cone-associated protein (PalmtdTomato). The plasmid was kindly provided by Dr Bakhos Tannous (Massachusetts General Hospital, Boston, MA, USA). The harvested supernatants were collected to isolate exosomes according to a previous study [53 ]. The supernatant was centrifuged at 1000g for 30 min followed by 10,000g for 30 min at 4 °C to remove cells and debris and then was centrifuged at 140,000g for 90 min at 4 °C in a Type Ti70 rotor using an L-80XP ultracentrifuge (Beckman). After resuspension in PBS, the exosome pellet was ultracentrifuged again for 90 min at 140,000g. Finally, the exosomes were resuspended in PBS, filtered using a 0.22-μm filter (Millipore), and analyzed with a Micro BCA Protein Assay kit (Pierce).
3
Isolation and Purification of Muscle Cell-Derived Extracellular Vesicles
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EVs were isolated from conditioned medium of C2C12 myoblasts, differentiated into myotubes, using HS, previously centrifuged at 100,000× g for 16 h at 4 °C for EV depletion. After 48 h of incubation in fresh medium, EVs were harvested and purified by differential centrifugation—cell debris and organelles were eliminated at 500× g for 20 min followed by another centrifugation at 3500× g for 15 min at 4 °C. EVs were pelleted by ultracentrifugation at 100,000× g for 70 min at 4 °C by L-80-XP ultracentrifuge (Beckman-Coulter, Brea, CA, USA). Finally, the pellet was washed with cold PBS (Phosphate Buffered Saline) in order to minimize sticking and trapping of non-vesicular materials. Purified EVs were used immediately after isolation.
4
Exosome Isolation and Characterization
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Prior to cell culture, DMEM containing 20% FBS was centrifuged at 120,000 × g for 2 h to deplete serum exosomes. 293T cells, used for exosome production, were cultured in 30 mL of DMEM + 5% exosome-depleted FBS in a 150-mm dish and maintained in 5% CO2 at 37°C for 48 h. Exosomes were isolated from the 30-mL harvested supernatant according to a previous report.49 The filtrate was centrifuged at 110,000 × g for 120 min at 4°C in a type Ti70 rotor, using an L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA, USA). The exosome pellet was resuspended in PBS and ultracentrifuged again at 110,000 × g for 120 min. The pelleted exosomes were resuspended in PBS and analyzed using a Micro BCA (bicinchoninic acid) protein assay kit (Pierce, Rockford, IL, USA) or by western blot analysis of exosomal markers using antibodies specific for ALIX, Flotillin 1, GM130, and HA-tag (Proteintech, Wuhan, China).
5
EV Isolation from Raw264.7 Cell Culture
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Before culture, EVs were depleted through centrifugation at 120,000×g for 18 h using FBS. Raw264.7 cells were cultured with 10% EVs-depleted FBS. Approximately 1000 mL supernatant (5 × 108 cells for 48-h culture) was subjected to centrifugation in a 100 KDa ultrafiltration centrifuge tube. The retentate was re-suspended in 100 mL PBS, centrifuged at 300×g for 10 min at 4 °C, at 2000×g for 10 min, and at 10,000×g for 30 min to remove cells and debris. The supernatant was filtered through 0.22 µm filter, followed by centrifugation in Ti70 rotor of L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 120,000×g for 70 min at 4 °C. The pelleted EVs were re-suspended in PBS and ultra-centrifuged at 120,000×g for 70 min. The EVs were again re-suspended in PBS and stored at − 80 °C for further use. The protein concentration in EVs was measured using the bicinchoninic acid (BCA) protein detection kit (Thermo Fisher Scientific, Santa Clara, CA, USA).
6
Extracellular Vesicle Isolation Workflow
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ReN cells were cultured for 72 h and the supernatant was collected. The supernatant was centrifuged at 300 g for 10 min, 1200 g for 20 min, and 10,000 g for 30 min at 4 °C to remove cells and debris and then filtered using a 0.22-μm filter (Millipore). The filtrate was centrifuged at 200,000 g for 90 min at 4 °C in a Type Ti70 rotor using an L-80XP ultracentrifuge (Beckman Coulter, Brea, USA). The pellet was resuspended in PBS and ultracentrifuged again at 200,000 g for 90 min. The EV pellets were resuspended with double-0.22 μm-filtered PBS. The protein concentration was determined by BCA protein assay (Pierce, Rockford, IL, USA). Western blotting was performed with anti-Alix, anti-TSG101, and anti-Calnexin antibodies (Abcam, Cambridge, UK) to analyze EV markers and negative marker. For EV293 isolation, HEK293T cells were cultured in DMEM with 10% EV-depleted FBS for 72 h. The supernatant was collected and conducted to differential centrifugation as described above.
7
Comparative SILAC Proteomics of BxPC3 Cells
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BxPC3 and BxPC3-SMAD4+ cell lines were cultured in RPMI 1640 MEDIA FOR SILAC with 10% dialyzed fetal bovine serum (FBS), with the addition of either with the non-labelled aminoacids Lysin and Arginine (light medium) or with the labelled 13C6-Lysine and 13C615N4-arginine (heavy medium) (Chemical Research 2000 srl, Rome, Italy). In a second experiment, the same cell lines were cultured by swapping media, BxPC3 being maintained in heavy medium and BxPC3-SMAD4+ maintained in light medium, thus creating two biological replicates of the same experiment. After eight days, cell media were changed with fresh media prepared as specified above but without the serum addition, in order to reduce any contaminant in the proteomic analyses, cells being cultured in this condition for 16 hours before media collection and exosomes enrichment made by ultracentrifugation (Beckman Coulter L-80XP Ultracentrifuge, Type 70 Ti rotor) as detailed in supplementary methods . The ratios for light/heavy and heavy/light were calculated for each identified protein of the two experiments. Proteins were considered significantly altered if the average value, calculated for the two ratios, was above 1.5 or below 0.667
8
Esophageal Cancer Cell Line Culture
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The human esophageal cancer cell line EC9706 (National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) was cultured in RPMI-1640 (Gibco, USA) medium. HUVECs (Shanghai Institute of Biochemistry and Cell Biology, CAS) were cultured in F12K medium (Gibco, USA). Both of the basal culture media above were supplemented with 10% fetal bovine serum (FBS, Gibco, USA)and 1% penicillin and streptomycin. FBS was centrifuged 10,000× g for 30 min, followed by ultracentrifugation at 200,000×g for 6 h to eliminate bovine-derived exosomes using a Type 70 Ti rotor in L-80XP ultracentrifuge (Beckman Coulter, Brea, Ca, USA). Cells were incubated in a humidi ed incubator at 37˚C with 5% CO 2 .
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