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).
Flotillin 1
Manufactured by Proteintech
Sourced in United States
Flotillin 1 is a protein that functions as a lipid raft-associated protein, involved in the organization of microdomains in the cell membrane. It plays a role in processes such as endocytosis and cell signaling.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (2 mentions)
Caveolin 1, by Proteintech (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
L 80xp ultracentrifuge, by Beckman Coulter (1 mentions)
Ha tag, by Proteintech (1 mentions)
Calnexin, by Santa Cruz Biotechnology (1 mentions)
Azure 600 system, by Azure Biosystems (1 mentions)
Gm130, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Cytochrome c, by Santa Cruz Biotechnology (1 mentions)
Odyssey 9120, by LI COR (1 mentions)
Psen1, by Cell Signaling Technology (1 mentions)
Criterion cell, by Bio-Rad (1 mentions)
Revert 700, by LI COR (1 mentions)
βiii tubulin, by Proteintech (1 mentions)
Psd95, by Proteintech (1 mentions)
7 protocols using flotillin 1
1
Exosome Isolation and Characterization
2
Comprehensive EV Protein Analysis Protocol
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Equal numbers of brain-derived EVs and the corresponding brain tissue lysate (normalized to protein concentration) were solubilized with 5 × Laemmli sample buffer (10% SDS, 1 mg/mL bromophenol blue, 250 mM Tris pH 6.8, 5% β-mercaptoethanol, 0.5 M DTT, 50% glycerol), followed by heating to 95 °C for 8 min. Protein separation was performed using pre-cast gels 4%–20% (mini-PROTEAN TGX, Biorad, Hercules, CA, USA) or SDS-PAGE gels (8% or 10%) and transferred to nitrocellulose membranes. Nonspecific binding was inhibited by blocking with 5% NFDM (Blotting-grade blocker, Biorad) for 1 h. Blots were then probed with primary antibodies overnight at 4 °C and incubated with an HRP-conjugated secondary antibody. Either SuperSignal™ West Femto Substrate (ThermoFisher, Waltham, MA, USA) or Clarity Western ECL Substrate (Biorad) were used for membrane development, and images were acquired on a ChemiDoc imaging system (Biorad) or Azure 600 system (Azure Biosystems, Dublin, CA, USA). Primary antibodies used were CD9, CD63 (ExoAb Antibody Kit, Systems Biociences, Palo Alto, CA, USA) at a dilution of 1:1000, GM130 (sc-55591, Santa Cruz Biotechnology, Dallas, TX, USA) at 1:200 dilution, ASM at 1:500 dilution (14609-1-AP, Proteintech), Flotillin 1 (15571-1-AP, Proteintech, Rosemont, IL, USA), Calnexin (sc-46669, Santa Cruz) at 1:200, and Alix (sc-53540, Santa Cruz) at 1:100.
3
Characterization of Placental Extracellular Vesicles
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BeWo cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Massachusetts). Cell lysates were used as a positive control and as tissue confirmation of the protein markers being assessed. Equal quantities of EV protein and cell lysate (10 µg) were run on polyacrylamide gels and then transferred onto nitrocellulose membranes (Life Technologies, New York). After blocking with 5% milk, membranes were probed with primary antibodies specific to the following proteins: syncytin-1 (1:500, Santa Cruz Biotechnology, California), PLAP (1:200, Santa Cruz Biotechnology, California), Plac-1 (1:200, Santa Cruz Biotechnology, California), CD63 (1:1000, Santa Cruz Biotechnology, California), Cytochrome C (1:500, Santa Cruz Biotechnology, California), β-actin (1:200, Proteintech, Illinois), and flotillin-1 (1:1000, Proteintech, Illinois). Horseradish peroxidase coupled secondary antibodies were added and detected through chemiluminescence using ImageQuant LAS 400 phosphoimager.
4
Western Blot Analysis of Neurodegeneration Markers
5
Comprehensive Extracellular Vesicle Protein Analysis
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Purified sEV samples were incubated on ice in 10X RIPA buffer supplemented with protease inhibitor cocktail (Cell Signaling Technology, #9806) and proteosome inhibitor MG-132 (Sigma, #M7449). Either 4X reducing sample buffer or 4X LDS non-reducing sample buffer (Thermo Scientific, #84,788) was added to the sEVs before heating at 95°C for five minutes. sEV samples were electrophoresed on SDS-polyacrylamide gels and subsequently transferred to a methanol-activated Immobilon-P Transfer Membrane (#IPVH00010 Immobilon). After blocking at room temperature for one hour with blocking buffer (1X TBS pH 7.6 containing 0.05% Tween-20 and 5% fat-free dry milk), membranes were incubated at 4°C with rocking overnight using primary antibodies for the following proteins: ALIX (1/1,000, Proteintech #67,715-1-Ig), β-Actin (1/10,000, Santa Cruz Biotechnology, #sc-47778), CD-9 (1/1,000, Proteintech #20,597-1-AP), Flotillin 1 (1/1,000, Proteintech #15,571-1-AP), GAPDH (1/2,000, Cell Signaling Technology #5174), Hif-1α (1/1,000, Novus Biologicals #NB100-449), placental alkaline phosphatase (1/1,000, Boster Biological Technology #A01718), TSG101 (1/1,000, Proteintech #28,283-1-AP). Following incubation, membranes were subsequently washed and incubated with secondary antibody at 1/10,000 for 1 h at room temperature and visualized via West Pico SuperSignal chemiluminescence.
6
Exosome Purification and Characterization Protocol
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Cell culture medium was centrifuged at 500 g for 10 min, then the supernatant was centrifuged at 20 000 g for 20 min. The supernatant was transferred to a fresh tube, filtered through a 0.22 µm filter and pelleted by ultracentrifugation (Beckman Optima L‐100 XP, Beckman Coulter) at 100 000 g for 70 min. Exosome pellets were washed in a large volume of PBS and recovered by centrifugation at 100 000 g for 70 min. The particle concentration and size distribution of the purified exosomes were determined using NTA in ZetaView (Particle Metrix). The morphology and size of the exosomes were determined using SEM. Purified exosomes were characterized by western blotting using antibodies against TSG101 (Abcam, ab125011), CD9 (System Biosciences, ExoAB‐CD9A‐1), Calnexin (Cell Signaling Technology, 2679), Flotillin1 (Proteintech, 15571‐1), and CD81 (Proteintech, 66866‐1).
7
Western Blot Analysis of Cell Signaling
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Western blots were performed in the same way as previously described (Zhang et al. 2021 (link)). GAPDH (1:10,000 dilution), β-Actin (1:3,000 dilution), EGFR (1:1,000 dilution), Caveolin-1 (CAV1, 1:1,000 dilution), Flotillin 1 (FLOT1, 1:2,000 dilution), Desmoplakin (DSP, 1:1,000 dilution), E-cadherin (E-CAD, 1:2,0000 dilution), N-cadherin (N-CAD, 1:4,000 dilution), Vimentin (Vim, 1:4,000 dilution), STAT3 (signal transducer and activator of transcription, 1:3,000 dilution), Erk (extracellular regulated protein kinases, 1:3,000 dilution), and p-Erk (1:3,000 dilution) were purchased from Proteintech. AKT (protein kinase B, 1:1,000 dilution) and p-AKT (1:1,000 dilution) were purchased from Abcam.
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