Cells were seeded at a 100 mm dish and cultured for 48 h under NG or HG condition in culture medium containing 1% exosome-free FBS (Life Technologies). The RNAs of exosomes derived from the supernatants of cells and the urine were purified using ExoQuick-TC and urine Exosome RNA Isolation Kit (NORGEN, Canada) following the manufacturer’s protocols. Exosome size was determined using nanoparticle tracking analysis [15 (link)].
Exosome free fbs
Manufactured by Thermo Fisher Scientific
Sourced in United States
Exosome-free FBS is a specialized cell culture supplement produced by Thermo Fisher Scientific. It is designed to provide a serum-based growth environment for cells, while minimizing the presence of contaminating exosomes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Urine exosome rna isolation kit, by Norgen Biotek (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Penicillin streptomycin solution, by Corning (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Universal mycoplasma detection kit, by American Type Culture Collection (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Fluoroblok insert, by Corning (1 mentions)
Hbmec, by Merck Group (1 mentions)
Gw4869, by Merck Group (1 mentions)
Tnf α, by R&D Systems (1 mentions)
70kd fitc dextran, by Merck Group (1 mentions)
Dnase rnase free water, by Thermo Fisher Scientific (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Eurobio Scientific (1 mentions)
Inorganic phosphate, by Merck Group (1 mentions)
26 protocols using exosome free fbs
1
Isolation and Characterization of Exosomes
2
Isolation and Characterization of Extracellular Vesicles
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Cells were grown in ATCC recommended media in 5% CO2 at 37°C. After reaching 90% confluence, the medium was replaced with medium containing 5% exosome‐free FBS (Life Technology Inc.). After 24 h, the conditioned medium was collected for isolation of the EVs. Specifically, supernatants were collected after a series of centrifugations at 300 × g at 4°C for 10 min, 2000 × g at 4°C for 10 min, and 10,000 × g at 4°C for 30 min to remove live cells, dead cells, and cell debris, respectively. The supernatant was finally subjected to ultracentrifugation at 100,000 × g at 4°C for 90 min. The EVs pellet was washed in 1X PBS, and ultracentrifuged at 100,000 × g at 4°C for 90 min. The pelleted EVs were re‐suspended either in 1X PBS for EVs characterization or lysed in RIPA buffer for protein analysis.
3
Osteoblast-Osteoclast Co-culture Protocol
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The well inserts with a 0.4-μm pore-sized filter (Greiner) for six-well plates were used following the manufacturer's instructions. Primary osteoblast precursor cells were seeded into the well inserts and differentiated into osteoblasts with osteogenic α-MEM medium. BMMs were seeded into six-well plates and induced into osteoclasts by complete α–MEM containing M–CSF and RANKL. After differentiation, osteoblasts and osteoclasts were washed with PBS, and then co-cultured for different time according to the experimental requirements. All co-culture experiments were done in complete α-MEM with exosome-free FBS (Life Technologies). The lentiviral vector expressing ATF4 3′UTR (ATF4 3′UTR)/Negative control lentivirus (3′UTR NC), lentiviral vector encoding red fluorescence protein (LV-RFP) and CMV-GFP-CD63 lentiviral vector (GFP-CD63) were purchased from Shanghai Integrated Biotech Solutions Co., Ltd. In the case of ATF4 3′UTR (3′UTR NC)/GFP-CD63-transfected osteoclasts and LV-REP-transfected osteoblasts, the cells were transfected at a multiplicity of infection (MOI) of 100 the day before the co-culture experiment according to the manufacturer's instructions.
4
Cell Line Culture and Characterization
5
Transendothelial Migration Assay
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Human brain microvascular endothelial cells (HBMECs, Cell Systems, Kirkland, WA, USA) were cultured in complete endothelial cell medium (Cell Systems) and used for experiments up to passage 9. Cells grown to 80% confluency in 24-well plates were used for the coculture assay. On the day of experiment, the medium was changed to complete recombinant serum free medium with 10% exosome free FBS (Cell Systems). Nonstimulated (NS) or activated monocytes were stained with calcein AM (1.5 μM) (Thermo Fisher Scientific) for 30 min at 37 °C, washed with PBS and re-suspended in RPMI containing 10% exosome-free FBS. They were seeded at a concentration of 4 × 105 cells per FluoroBlokTM insert of 8 μm pore size (Corning, Corning, Coring, NY, USA). The inserts were placed on top of the confluent HBMECs and exosome inhibitor GW4869 (10 μM)44 (link) (Sigma-Aldrich) was immediately added in each insert per stimulation group (NS, IFNα, LPS, I/L). The monocyte migration toward HBMECs was quantified by measuring the increase in fluorescence at 485 nm excitation and 538 nm emission rate in the bottom chamber after 3 h of incubation at 37 °C.
6
Leukocyte Endothelial Cell Culture and Permeability
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Early passage LECs were cultured to confluence in polycarbonate six-well transwell cell culture inserts (0.4-µm pore size, 108 pores/cm2; Corning) in EGM2MV media. Leakage was tested on monolayers with 70-kD FITC-dextran (10 µg/ml; Sigma-Aldrich). Maximal permeability was determined without cells. Basolateral LEC culture supernatants were collected after 24 h of culturing in EGM2MV media that was supplemented with 10% exosome-free FBS (Thermo Fisher Scientific) instead of 10% FBS, which is contaminated by bovine-derived exosomes as provided by the manufacturer. In some experiments LECs were incubated in exosome-free EGM2MV media containing 7 ng/ml TNF-α (R&D Systems).
7
Cellular Oxidative Stress Assays
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Fetal bovine serum (FBS) and glutamine were purchased from Eurobio® (Les Ulis, France). Lipopolysaccharide from Escherichia coli K12 (LPS-EK) was obtained from InvivoGen® (San Diego, California, United States) and inorganic phosphate (Pi) from Merck® (Darmstadt, Germany). Exosome-free FBS, TRIzol™ Reagent, RNase/DNase-free water, High-Capacity RNA-to-cDNA™ kits, BCA™ Protein Assay kits, and dihydroethidium (DHE) were purchased from Thermo Fisher Scientific® (Waltham, Massachusetts, United States). Takyon™ was obtained from Eurogentec® (Liège, Belgium). 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes® (Eugene, Oregon, United States). All other molecular and biochemical reagents were obtained from Sigma-Aldrich® (Saint-Louis, Missouri, United States).
8
Exosome Isolation and Characterization
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Cells were seeded at densities of 3–5 × 105/mL in complete media with 10% exosome free‐FBS (Thermofisher) and supernatants were collected at day 5. Differential centrifugation at 400×g and 2000×g for 10 min each was done to remove cells and debris. The resulting supernatant was either directly centrifuged or first mixed with the Total Exosome Isolation reagent (Invitrogen) at a ratio of 1:2 overnight at 4°C and centrifuged at 3000 RPM for 5 h to collect all EVs. For exosomes, EV pellets were resuspended and washed in 2 mL DPBS and ultracentrifuged at 27,000 RPM (100,000×g) for 2.5–3 h at 4°C. Isolated exosomes were resuspended in DPBS at a desired volume and subjected to protein quantification by the BCA protein assay (Pierce).
For EV fractionation studies, pellets collected after centrifugation at 2000×g are denoted 2k, 1000×g denoted 10k, and 100,000×g denoted 100k. EV validation and characterization was performed by Western blotting, Nanoparticle Tracking Analysis (NTA), and by a newer technology known as ExoView/NanoView, further described below.
For EV fractionation studies, pellets collected after centrifugation at 2000×g are denoted 2k, 1000×g denoted 10k, and 100,000×g denoted 100k. EV validation and characterization was performed by Western blotting, Nanoparticle Tracking Analysis (NTA), and by a newer technology known as ExoView/NanoView, further described below.
9
Isolation of Exosomes from NPc Cells
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Serial centrifugation with ultracentrifugation was used to isolate dNPc-exo and nNPc-exo. Briefly, non-degenerated and degenerated NPc were inoculated with 1 × 106 cells and grown in basal medium of DMEM-F12. When the cells reached 80% confluence, exosome-free FBS (Thermo Fisher Scientific, Waltham, MA, USA) was used to culture cells. After 48 h, collected the medium and centrifuged at 300×g (4 °C) for 10 min to remove dead cells. Then, obtained the supernatant and centrifuged at 2000×g (4 °C) for 20 min to remove cell debris. Next, the supernatant was centrifuged at 10,000×g (4 °C) for 30 min to remove large EVs. To remove substructures and vesicles larger than 220 nm, we filtered the supernatant through a 0.22-µm filter (Millipore, US). Then, pelleted the exosomes using ultracentrifugation at 100,000×g for 80 min (4 °C). After resuspending in sterile PBS, the exosomes were stored at -80 °C.
10
Extracellular Vesicle Isolation from MOVAS-1 Cells
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MOVAS-1 cells were seeded in 10-cm Petri plates (600,000 cells/Petri plate), cultured in DMEM 6546 medium supplemented with 10% FCS for 2 days and then treated with 1% exosome-free FBS (ThermoFisher Scientific, New York, NY, United States) for 10 days. EVs were extracted from culture supernatants by sequential centrifugations as described previously by Théry et al. (2006) . Briefly, cells and dead cells were first removed by 10-min centrifugations at 300 × g and 2,000 × g. Then, the collected supernatant was centrifuged at 10,000 × g for 30 min to get rid of large cellular debris. After centrifugation, the supernatant was recovered and centrifuged at 100,000 × g for 70 min. The pellet was then resuspended in cold PBS and centrifuged at 100,000 × g for another 70 min. The obtained pellet which contains EVs was resuspended in PBS then immediately stored at -80°C for future analysis. The pellets’ protein concentrations were determined with a BCA Protein Assay Reagent Kit (ThermoFisher Scientific, Rockford, IL, United States).
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