The rat HERS cell line used in this study was established in our previous study 35 (link). HERS-H1 cells were cultured in epithelial medium (ScienCell, USA) supplemented with 1% penicillin/streptomycin. To collect the culture medium for isolation of ELVs, HERS-H1 cells were cultured to 80% confluence, washed twice with PBS, then switched to medium with exosome-depleted FBS (2%, SBI, USA) and cultured for another 2 d. Dental papilla cells (DPCs) were isolated from unerupted first molars of 1 to 3-d-old postnatal SD rats as previously described 37 . To inhibit the activity of the Wnt/β-catenin pathway, dickkopf WNT signaling pathway inhibitor 1 (DKK1) (100 ng/mL, R&D systems, USA) was used for 2 h to induce the phosphorylated degradation of β-catenin. Cells were grown in a humidified atmosphere at 37 °C with 5 % CO2, and the medium was changed every 3 d.
Fetal bovine serum (fbs)
Manufactured by System Biosciences
Sourced in United States, Japan
Fetal Bovine Serum (FBS) is a common cell culture supplement derived from the blood of bovine fetuses. It provides a rich source of growth factors, nutrients, and other components that support the growth and proliferation of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
0.22 μm filter, by Merck Group (3 mentions)
Rpmi 1640, by System Biosciences (2 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (2 mentions)
Met 5a, by American Type Culture Collection (2 mentions)
Wi 38, by Nacalai Tesque (2 mentions)
Met 5a, by Nacalai Tesque (2 mentions)
H 7650, by Hitachi (2 mentions)
Human umbilical vein endothelial cells (huvec), by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Ht 29, by American Type Culture Collection (2 mentions)
Gw4869, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by System Biosciences (2 mentions)
Streptomycin, by System Biosciences (2 mentions)
Exorneasy midi kit, by Qiagen (1 mentions)
Optima max xp, by Beckman Coulter (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Nanosight lm10, by Malvern Panalytical (1 mentions)
53 protocols using fetal bovine serum (fbs)
1
Rat HERS Cell Line and DPC Isolation
2
Cell Line Culture for Leukemia and Lung Cancer
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Human acute monocytic leukemia (THP-1) and human lung cancer (A549) cell lines were purchased from the cell bank of Shanghai institute of life sciences (Shanghai, China). THP-1 and A549 cell lines were cultured by using Roswell Park Memorial Institute 1640 (RPMI-1640) medium added with 10% exosomes-depleted FBS (SBI, Johnston, Pennsylvania, USA).18 (link) All cells were cultured in an incubator (37 °C) with humidified environment and CO2 (5%).
3
Exosome and miRNA Extraction from ADSCs
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ADSCs were cultured in DMEM containing 10% exosomes-free FBS (180625; SBI) for 48 h. The medium was then collected and centrifuged at 3000 × g for 10 min at 4°C. The supernatant was collected, transferred to a new tube, and placed on ice. Exosomes and miRNAs were extracted using an exoRNeasy Midi Kit (77144; QIAGEN), according to the manufacturer's protocol.
4
Exosome Isolation from Cell Culture
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The cells were cultured in a medium supplemented with 2% exosome depleted FBS (SBI, USA). Centrifuge cultural supernatants for 10 minutes at 1500 g to remove dead cells. After discarding the pellet, the supernatant was transferred to a new collection tube. Then the supernatant was ultra-centrifuged at 12 000 g for 30 minutes (Beckman Coulter Optima MAX-XP), and exosomes were pelleted by ultracentrifugation at 100 000 g for 70 minutes. After discarding the supernatant, the exosome pellet was re-suspended in phosphate-buffered saline (PBS) and centrifuged at 100 000 g for 70 hours.
5
Transwell Migration Assay for Exosome Effects
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transwell migration assay was used to measure the effect of IONP-Exos and BMSC-Exos on NIH3T3 fibroblast migration and was performed according to the manufacturer's recommendation. Briefly, a total of 5 × 104 NIH3T3 fibroblasts were seeded in the upper chamber of the transwell (Corning, NY, USA) with 100 μL of serum-free medium. In the lower chamber, 600 μL of culture medium containing 10% exosome-depleted FBS (SBI, USA) and IONP-Exos (100 μg/mL), BMSC-Exos (100 μg/mL), or the equivalent volume of exosome diluent (PBS) were added and incubated for 24 h. Fibroblasts on the permeable membrane of the upper chamber were gently wiped off using a cotton swab, and fibroblasts that migrated to the bottom of the filter membrane were fixed with anhydrous ethanol and stained with crystal violet (Beyotime, Shanghai, China). Five fields of the underside of the membrane were randomly selected, and the migrated cells were counted under a microscope.
6
Isolation and Characterization of MSC-Derived Extracellular Vesicles
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Synovial MSCs were seeded at 1.5 × 106 cells per 15-cm2 plate and incubated in a complete culture medium for 24 h. The cells were then washed twice with phosphate-buffered saline (PBS) and cultured with alpha MEM containing 5% exosome-depleted FBS (SBI, Palo Alto, CA, USA) or serum-free alpha MEM for 48 h. The cell suspension was centrifuged at 2000×g and 4 °C for 10 min to remove detached cells. The supernatant was collected and passed through a 0.22-μm filter (EMD Millipore, Billerica, MA, USA) to remove cellular debris. The filtered supernatant was then transferred to the upper compartment of a 10-kDa Amicon Ultra Filter Unit (EMD Millipore, Billerica, MA, USA) and centrifuged at 4000×g and 4 °C for 30 min. The liquid was washed with PBS and subjected to ultrafiltration. The MSC-EVs were collected from the upper compartment and stored in a PROKEEP low-protein binding tube (WATSON, Tokyo, Japan) at − 80 °C. Nanoparticle tracking analysis (NTA) was performed on the EVs using NanoSight LM10 and NanoSight NTA v3.0 (Malvern Panalytical, Malvern, UK). Western blotting was used to identify the EV-associated markers CD9 and CD63. MSC-EVs were negatively stained with 2% (w/v) uranyl acetate, and their morphology was observed under a transmission electron microscope (TEM; JEM-1400 Flash; JEOL Ltd., Tokyo, Japan).
7
Isolation and Activation of Human Neutrophils
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PMNs were isolated from human peripheral blood using the EasySep™ direct human neutrophil isolation kit (Stemcell, Vancouver, Canada). PMNs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Waltham, USA) supplemented with 10% exosome-free fetal bovine serum (FBS) (SBI, California, USA) and 1% penicillin and streptomycin (Beyotime, Shanghai, China) in a 37 °C incubator with a 5% CO2 air atmosphere. The lipopolysaccharide (LPS; Sigma, Saint Louis, USA) concentration of the activated PMNs (aPMNs) was 1 µg/mL for 30 min.
8
Exosome Isolation by Differential Centrifugation
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Exosomes were isolated by differential centrifugation of conditioned media collected from TSGH-8301, T24 and J82 cells. Cells were grown in medium containing 10% exosome-depleted FBS (SBI System Biosciences, Palo Alto, CA, USA). After removing cells and other debris by centrifugation at 3000 × g for 30 minutes, the supernatant was subsequently centrifuged at 10000 × g for 1 hour to remove shedding vesicles and other large vesicles. Finally, the supernatant was recentrifuged at 120000 × g for 3 hours at 4 °C. The exosome pellets were resuspended in PBS and stored at 4 °C before experimental analyses.
9
Isolation of Exosomes from Cell Supernatants
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The exosomes from the MSC (MSC‐Exo), HepG2 (HCC‐Exo), and SW1116 (CRC‐Exo) cell supernatant were isolated through the density gradient ultracentrifugation method according to relevant literatures.
32 (link),
33 (link) Briefly, 2 × 107 cells were cultured in 10 mL DMEM with 10% exosome‐free FBS (System Biosciences, Palo Alto, CA, USA). After culturing for 48 h until the cell reached 80% confluence, 210 mL medium was collected into 50 mL. Centrifuge cells at 4 °C, 300 g for 10 min, 2000 g for 10 min, and then 10,000 g for 30 min to remove adherent cells, dead cells, and cell debris. Then, the supernatant was removed to a new 50 mL tube and centrifuged at 4 °C, 100000 g for 70 min to collect exosomes. Next, the supernatant was carefully removed, and the pellet was re‐suspended with 35 mL PBS to wash the exosomes. Finally, the sample was centrifuged for another 70 min at 4 °C, 100000 g, and the pellet was re‐suspended with 1 × PBS (50 μL) and stored at −80°C.
32 (link),
33 (link) Briefly, 2 × 107 cells were cultured in 10 mL DMEM with 10% exosome‐free FBS (System Biosciences, Palo Alto, CA, USA). After culturing for 48 h until the cell reached 80% confluence, 210 mL medium was collected into 50 mL. Centrifuge cells at 4 °C, 300 g for 10 min, 2000 g for 10 min, and then 10,000 g for 30 min to remove adherent cells, dead cells, and cell debris. Then, the supernatant was removed to a new 50 mL tube and centrifuged at 4 °C, 100000 g for 70 min to collect exosomes. Next, the supernatant was carefully removed, and the pellet was re‐suspended with 35 mL PBS to wash the exosomes. Finally, the sample was centrifuged for another 70 min at 4 °C, 100000 g, and the pellet was re‐suspended with 1 × PBS (50 μL) and stored at −80°C.
10
Purification of Extracellular Vesicles from Transfected Cells
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HEK-293T cells were transfected with S-FLAG verctor, RBD-VSVG vector, CD9-GFP vector or empty vector using Lipofectamine 3000 when reached 70%–80% confluence. At 6 h after transfection, cells were switched to DMEM supplemented with 10% EV-depleted FBS(System Biosciences lnc) for the production of EVs. After 48 h, the cell supernatants was collected and centrifuged at 10,000g for 30 min, then subjected to filter twice through 0.22 μm membranes(Millpore). The EV pellets were prepared from filtered supernatants by ultracentrifugation at 100,000 g for 70 min at 4 °C (Beckman Ti70 rotor). Resuspend the pellets in phosphate buffered saline (PBS) and repeat centrifuged 70 min at 100,000g at 4 °C to remove soluble serum and secreted proteins. The purified EV pellets were resuspended in fresh PBS(0.22 μm filtration).
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