SACC-83 cells (5×106) were seeded and cultured in T-75 cm2 flask with DMEM complete medium until they reach 80% confluence and further grown for 24 h in serum-free medium. Cell conditioned medium was then collected to isolate exosomes using the Total Exosome Isolation Reagent kit (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 4478359). Briefly, conditioned medium was centrifuged at 2,000 × g for 30 min at 4°C to remove cells and debris, and supernatant was collected, mixed with 5 ml Total Exosome Isolation Reagent and incubated at 4°C overnight. The sample was centrifuged at 10,000 × g for 1 h at 4°C and the subsequent exosome pellet was resuspended in 100 µl PBS. The exosomes from five flasks of cells were pooled. The size and particle concentration of exosomes were then determined with the ZetaView nanoparticle tracking analysis (NTA; Particle Metrix Inc.). Exosomes were quantified with a BCA Protein Assay kit (Thermo Scientific) using an Absorbance Reader (ELX-800, BioTek Instruments, Inc.), labeled with PKH67 Green Fluorescent Cell Linker (Sigma-Aldrich; Merck KGaA; cat. no. PKH67GL) according to the manufacturer's protocol.
Total exosome isolation reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Total Exosome Isolation Reagent Kit is a laboratory product designed for the isolation of exosomes from various sample types, including cell culture media and biological fluids. The kit utilizes a proprietary precipitation-based method to separate exosomes from other components in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pkh67 green fluorescent cell linker, by Merck Group (1 mentions)
Anti ve cadherin, by Abcam (1 mentions)
Fv1000 confocal laser microscope, by Olympus (1 mentions)
Anti rabbit fluorescent secondary antibody, by Proteintech (1 mentions)
Glass bottom cell culture dish, by NEST Biotechnology (1 mentions)
Transmission electron microscopy, by Thermo Fisher Scientific (1 mentions)
Total exosome rna and protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
12 protocols using total exosome isolation reagent kit
1
Isolation and Characterization of Exosomes
2
Exosome Isolation and Tregs Incubation
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Cells were grown to the third generation in RPMI 1640 medium without exosomes. The supernatant of culture was collected by centrifugation at 3000g for 15 min to remove cell debris, and then filtrated by 0.22 µm ultrafiltrate membrane under aseptic conditions. Next, the liquid was concentrated by ultrafiltration tube. The exosomes were extracted by Total Exosome Isolation Reagent Kit (from cell culture media) (Invitrogen) as the introduction described. Then, the exosomes were incubated with Tregs.
3
Exosome Isolation from Cell Culture
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After the cells were cultured in 10% FBS (EV-free) medium for 48 h, we harvested the culture supernatant. Afterwards, the exosomes were extracted using a total exosome isolation reagent kit (Invitrogen) in accordance with the instructions of the manufacture. Subsequently, we resuspended the exosomes in PBS.
4
Exosome Isolation from Conditioned Media
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Free cell CM samples were used for exosome isolation using the Total Exosome Isolation Reagent Kit (Invitrogen, CA, USA). Briefly, 2 ml exosome isolation reagent was added to 4 ml of CM, incubated 12 h at 4°C, and centrifuged at 10000 × g for 1 h to obtain pelleted exosomes and non-exosome supernatant. The exosome pellets were suspended in 100 μl of PBS. Total RNA and proteins in the exosomes and exosome-depleted supernatant were isolated for miRNA PCR analysis and marker protein detection, respectively.
5
Exosome Labeling and Immunofluorescence Imaging
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Cells were seeded in the glass-bottom cell culture dish (NEST, San Diego, CA, USA). Exosomes were labeled with Dio (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, exosomes were incubated for 30 min with Dio at a final concentration of 10 μM at 37 °C and re-isolated using the Total Exosome Isolation Reagent Kit (Invitrogen). After being treated with 4% paraformaldehyde, the cells were permeabilized with 0.01% Triton X-100 for 10 min, and then treated with anti-VE-cadherin (Abcam) or anti-ZO-1 monoclonal antibody (Abcam). After being washed, the cells were treated with anti-rabbit fluorescent secondary antibody (Proteintech, Wuhan, China) and fluorescence was observed and photographed using an Olympus FV1000 laser confocal microscope.
6
Isolation and Characterization of Exosomes
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Exosomes were isolated using a Total Exosome Isolation Reagent Kit (Invitrogen). After the attachment of HCT116 cells, culture mediums in 10 cm dishes were replaced by serum‐free cell culture medium with or without YQ456. After incubation for 24 h, cells were harvested and centrifuged at 2500 × g for 25 min. The supernatant was added to the exosome precipitant and incubated overnight at 4°C. The mixture was centrifuged at 11 000 × g for 1 h at 4°C. Exosome pellets were resuspended in 50 μL PBS.
7
Exosome Isolation from Sera and BAL Fluid
8
Exosome Isolation from Treg Cell Culture
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Tregs were cultured in RPMI-1640 medium without exosomes. After 48 hr of cultivation, the supernatant of culture was collected by centrifugation, and then filtrated by 0.22 μm ultrafiltrate membrane. Next, the liquid was concentrated by ultrafiltration tube. The exosomes was extracted by Total exosome isolation reagent kit (from cell culture media) (Invitrogen) following the manufacturer's protocol. The transmission electron microscopy (Thermo Fisher Scientific) was used to analyze the ultrastructure of exosomes. Western blot (WB) was performed to detect the expression of exosomes markers, including TSG101 and CD63.
9
Isolation and Characterization of Extracellular Vesicles
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EVs extraction from cell culture media was performed using a Total Exosome Isolation Reagent Kit (Thermo Fisher Scientific Inc., Waltham, MA USA) with some modifications. The dish with 70–80% confluent RAW264.7 cells was washed with 1 × PBS 3 times, serum-free medium was added, and the cells were cultured at 37 ℃ and 5% CO2 for 24 h. The medium was subsequently collected and centrifuged at 2000 g for 30 min followed by 10,000 g for 30 min. Then, the supernatant was collected and incubated with a half volume of EVs isolation reagent at 4 °C overnight. Finally, the mixture was centrifuged at 10,000 g for 1 h, and the precipitate was resuspended in PBS. EVs were detected using nanoparticle tracking analysis (NTA), transmission electron microscopy and western blotting or were stored at − 80 ℃.
For the in vitro experiment, VSMCs were treated with EVs at a ratio of 1:1,000 and cultured at 37 ℃ and 5.0% CO2 for 48 h. For the in vivo experiment, T2D mice were injected with 1.0 × 109 EVs via the tail vein once per week for two weeks.
For the in vitro experiment, VSMCs were treated with EVs at a ratio of 1:1,000 and cultured at 37 ℃ and 5.0% CO2 for 48 h. For the in vivo experiment, T2D mice were injected with 1.0 × 109 EVs via the tail vein once per week for two weeks.
10
Exosome Isolation from OS Cells
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Exosomes were isolated from the indicated OS cell lines and peripheral blood from OS patients using the Total Exosome Isolation Reagent kit (ThermoFisher Scientific, Carlsbad, CA, USA) strictly following the manufacturer’s protocol. Isolated exosomes were immediately processed for RNA and protein isolation.
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