All BMSC‐exosomes used in the in vitro and in vivo experiments were obtained from the same batch of BMSCs. FBS was ultracentrifuged at 120 000 × g under 4 °C for 12 h using an ultracentrifuge (Beckman Coulter) to prepare exosome‐free FBS. Exosome‐free culture medium was used to replace conventional culture medium as the cell density reached 80%. After the culture medium was gradient centrifuged to remove dead cells and cell debris, the supernatant was centrifuged at 100 000 × g for 90 min, and the isolated exosomes were resuspended in 50 µL PBS (Gibco) for further use. The NTA assay was used to detect the size of exosomes, and TEM (HT7700, HITACHI) was used to analyze the morphology of exosomes. WB was used to determine the expression of Flotillin‐1 (Abcam), TSG101 (Abcam), and CD63 (ProteinTech). A BCA protein assay kit (Thermo Fisher) was used to detect the concentration of exosomes.
Flotillin 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Flotillin-1 is a protein involved in the formation and organization of membrane microdomains known as lipid rafts. It is a component of these specialized membrane regions and plays a role in their assembly and stabilization.
Automatically generated - may contain errors
Lab products found in correlation
Tsg101, by Abcam (9 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
G box imaging system, by Syngene (3 mentions)
Tsg101, by BD (3 mentions)
β actin, by Merck Group (3 mentions)
Igg horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Actinin 4, by Gentex (2 mentions)
Mitofilin, by Thermo Fisher Scientific (2 mentions)
Lamp1, by Abcam (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Hsp70, by Abcam (2 mentions)
Ht7700 tem, by Hitachi (1 mentions)
Ultracentrifuge, by Beckman Coulter (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Transferrin receptor, by Thermo Fisher Scientific (1 mentions)
Nsmase2, by Santa Cruz Biotechnology (1 mentions)
Tecnai 12, by Philips (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Merck Group (1 mentions)
23 protocols using flotillin 1
1
Isolation and Characterization of BMSC-derived Exosomes
2
Western Blot Analysis of Extracellular Vesicle Markers
3
Exosome Characterization Using NanoFCM
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Exosome size was evaluated utilizing nanoparticle flow cytometry (NanoFCM; SNA-D1, UK), and NF Professional 1.0 software was used to explore their structural properties. Transmission electron microscopy (Tecnai 12, Philips) was employed to examine exosomal structure. The exosome-specific surface markers TSG101 (1:2000; Abcam, catalog no. ab125011), Flotillin-1 (1:2000; Abcam, catalog no. ab133497), Alix (1:2000; Abcam, catalog no. ab275377), and CD9 (1:1000; Cell Signaling Technology, catalog no. 98327) were evaluated utilizing western blot analysis.
4
Western Blot Analysis of Extracellular Vesicle Markers
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Briefly, extracted proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% milk incubated with the primary antibodies against the following: Alix (Bethyl Laboratories, A302-938A), Calretinin (Swant, CG1), CD63 (Santa Cruz Biotechnology, SC-15363), Flotillin1 (Abcam, AB41927), Porin (Calbiochem, 529536), Rab27a, TSG101 (Genetex, GTX70255), β-Actin (Sigma–Aldrich, A5441) overnight at 4°C, and finally incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). Protein bands were visualized using SuperSignal® West Pico Chemiluminescent Substrate (Millipore) and imaged using a Fuji-film LAS-3000/4000. Band densities were analyzed using ImageJ software. Protein levels were corrected to β-Actin. Protein data were analyzed and presented as for PCR data (see above).
5
Extracellular Vesicle Protein Analysis
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Antibodies against flotillin-1 (1 : 1000, Abcam), CD63 (1 : 500, Proteintech), GAPDH (1 : 500, Santa), VE-cadherin (1 : 1000, Abcam), CD9 (1 : 2000, CST), ALIX (1 : 1000, Abcam), TSG101 (1 : 1000, Abcam), and GM130 (1 : 1000, Abcam) were used in western blot as previously described [11 (link)].
6
Protein Quantification and Immunoblotting Analysis
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The protein amount in each fraction and pellet were measured by bicinchoninic acid protein assay. Equal amount of protein (20 μg) from each fraction and pellet were resolved by 10% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad; Hercules, CA). Nonspecific binding sites were blocked with 5% (w/v) milk in TBS containing 0.1% Tween 20 (TBS-T). After blocking, blots were incubated overnight with the primary polyclonal antibody flotillin 1 (1:1000; Abcam), CD63 (1:200; Santa Cruz Biotechnology), TSG101 (1:1000; BD Biosciences), actinin-4 (1:1000; Gentex), and mitofilin (1:5000; Thermo Fisher Scientific). After washes with TBS-T, blots were incubated for 2 h with the appropriate IgG horseradish peroxidase–linked secondary antibody (1:1000; Cell Signaling Technology) and developed by enhanced chemiluminescence. Image analysis was performed using a G: BOX imaging system (Syngene).
7
Extracellular Vesicle Profiling from Neuronal Cultures
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We used the Bio-Dot SF blotting apparatus from Bio-Rad to analyse media collected from neuronal cultures treated with ambroxol for 5 days. Before loading the media in the apparatus, debris and floating cells were removed by centrifugation. Bio-Dot SF blotting apparatus was assembled following the instruction manual and 100μl of media was loaded in the respective well. Dot blots were then washed with PBS and then blocked with Block Ace (BioRad) ON at 4 °C, followed by incubation with antibodies against CD63 (Santa Cruz Biotechnology, 1:100), Flotillin1 (Abcam, 1:1000), Lamp1 (Abcam 1:1000).
8
Western Blot Analysis of Exosomal Proteins
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After cell proteins were extracted, they were heated at 100 ℃ for 3 min to denature them. The protein was transferred to the negative control (NC) membrane, and the NC membrane was sealed in 5% skim milk powder [phosphate-buffered saline (PBS) preparation] at 37 ℃ for 2 h or 4 ℃ overnight. The primary antibody was then added to bind the target protein. The primary antibodies—CD9 (Abcam, Cambridge, UK), CD81 (Abcam, Cambridge, UK), Tumor susceptibility gene 101 (TSG101) (Abcam, Cambridge, UK), Heat Shock Protein 70 (HSP70) (Abcam, Cambridge, UK), ALG-2-interacting protein X (ALIX) (Abcam, Cambridge, UK), and flotillin-1 (Abcam, Cambridge, UK)—were incubated in a shaker at room temperature for 2 h or overnight at 4 ℃. The secondary antibody of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Abcam, Cambridge, UK) or goat anti-mouse (Abcam, Cambridge, UK) was incubated and incubated in a shaker for 1 h or overnight at 4 ℃. Diaminobenzidine (DAB) kit was used to develop color. ImageJ software (Bethesda, Maryland, USA) was used for gray value analysis of protein bands. Experimental results were recorded, and the NC film was dried, scanned, and preserved.
9
Protein Expression and Signaling Analysis
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Protein extraction and Western blots were conducted and analyzed as previously described [8 (link)] with the antibodies against the following: PTEN, N-Cad, and E-Cad (1:1000; Cell Signaling Technology, MA, USA); Akt and phosphorylated-Akt (phosphorylated on S473), Caspase 3 (1:1000; Abcam, CA, USA); P70S6K and phosphorylated-P70S6K (phosphorylated on S371), mTOR and phosphorylated-mTOR (phosphorylated on S2998; 1:1000; Bioworld, Jiangsu, China); CD63, TSG-101, and Flotillin-1 (1:500; Abcam, CA, USA), and human β-actin (1:5000; Transgene, Beijing, China). The blots were quantified by density relative to β-actin, while the phosphorylation of Akt, mTOR, and P70S6K was determined by density relative to total Akt, mTOR, and P70S6K, respectively. All experiments were performed in triplicate.
10
Immunofluorescence Analysis of Endocytic Markers
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HeLa cells were cultured overnight on glass coverslips and then treated with inhibitors or subjected to transfection. After being washed three times with PBS, the cells were fixed with ‐20°C prechilled methyl alcohol for 10 min and permeabilized with 0.1% Triton X‐100. After blocking with 5% BSA and 3% goat serum in PBS, the cells were incubated with primary antibodies (CD63, Invitrogen; HRS, Abcam; EEA1, abcam; LAMP1, abcam; Flotillin‐1, Abcam) overnight at 4°C in blocking buffer. The following day, after three washes in PBS, the cells were incubated with secondary antibodies (DyLight 488, DyLight 594, MultiSciences) for 30 min at RT, washed in PBS, and then mounted in antifade mounting medium with DAPI. The samples were imaged using an Olympus IX83‐FV3000 confocal microscope (Olympus). Images were analysed with ImageJ software.
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