Cultured ADSCs were plated at 5 × 105 cells in a 10-cm dish. The culture medium was collected and centrifuged at 13,000 g for 30 min to remove cells and cell debris. According to the manufacturer's instructions (System Biosciences, CA, United States), 2 mL of ExoQuick-TCTM exosome precipitation solution was used to isolate ADSC-Exo from 10 mL of culture medium. After overnight incubation at 4 °C, the mixture was centrifuged at 10,000 g for 30 min at 4 °C. After being washed, the exosomes were centrifuged at 10,000 g for 15 min at 4 °C. Suspended the purified ADSC-Exos with 100 µL PBS and stored at − 80 °C for further study. The protein extracted from the exosomes was quantified using the Bradford method. And then biomarker CD63 and CD9 were used to characterize the purified ADSC-Exos. The morphology of ADSC-Exo was detected through the transmission electron microscope (Hitachi, Tokyo, Japan). The size of ADSC-Exo was evaluated by Nanoparticle tracking analysis (NTA) analysis.
Exoquick tctm exosome precipitation solution
Manufactured by System Biosciences
Sourced in United States
ExoQuick-TCTM Exosome Precipitation Solution is a reagent designed to isolate and concentrate exosomes from biological samples, such as cell culture media or biofluids. It provides a simple and efficient method for the precipitation and recovery of extracellular vesicles, including exosomes, from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Exoquick exosome precipitation solution, by System Biosciences (3 mentions)
Transmission electron microscope, by Hitachi (2 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Ultrafilter, by Merck Group (1 mentions)
Tapi 2, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pkh26 labeling kit, by Merck Group (1 mentions)
7 protocols using exoquick tctm exosome precipitation solution
1
Isolation and Characterization of ADSC-Derived Exosomes
2
Isolation of Muscle-Derived Exosomes
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Fresh skeletal muscle tissues were collected from mice and immediately placed in vials containing Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen) supplemented with 10% Exosome-Depleted Fetal Bovine Serum (Exo-FBS; System Biosciences), 2% GlutaMAX (Invitrogen), and 1% Penicillin-Streptomycin (P-S; Invitrogen). The tissues were incubated in a humidified incubator set at 37°C and 5% CO2 for 24 h, allowing them to release extracellular vesicles, including muscle-derived exosomes, under conditions resembling the natural environment within the body. Medium was then collected and filtrated through 0.22 μm filters (Millipore) to remove cell debris and extracellular vesicles of a larger size. ExoQuick-TCTM Exosome Precipitation Solution (System Biosciences) was used to precipitate muscle-derived exosomes from the medium, according to manufacturer’s instructions. ExoQuickTM Exosome Precipitation Solution (System Biosciences) was used to isolate exosomes from serum samples following manufacturer’s instructions. In brief, appropriate amount of ExoQuick-TCTM or ExoQuickTM reagent was added to the filtered medium or serum samples, respectively, and incubated overnight at 4°C. The next day, the mixtures were centrifuged at 1,500 × g for 30 min and the exosome-containing pellet was collected and dissolved in the proper solution for further analysis.
3
Multiplex Gene Expression Analysis in Cultured Cells
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Total RNA from cultured cells was isolated using TRIzol reagent (Life Technologies). RNA from LCM was isolated using ExoQuick-TCTM exosome precipitation solution (System Biosciences, Mountain View, CA, USA). cDNA was synthesized from 500 ng of total RNA using PrimeScript reverse transcriptase (TAKARA, Shiga, Japan) and subjected to PCR. The following primer pairs were used for PCR: Gapdh, 5′-GTCAGTGGTGGACCTGACCT-3′ and 5′-AGGGGTCTACATGGCAACTG-3′ Alb, 5′-GTGCAAGAACTATGCTGAGG-3′ and 5′-ACTCACTGGGGTCTTCTCAT-3′ Ck18, 5′-GATTGACTGTGGAAGTGGATGC-3′ and 5′-GTTTGCATGGAGTTGCTGGA-3′ Afp, 5′-CACTGCTGCAACTCTTCGTA-3′and 5′-CTTTGGACCCTCTTCTGTGA-3′ Trop2, 5′-GTGTGCTGGTGCGTAAACTC-3′and 5′-ATGGTGGGCTCCTCATAGTG-3′ Dlk1, 5′-TGTCAATGGAGTCTGCAAGG-3′ and 5′-AGGGAGAACCATTGATCACG-3′ Sox9, 5′-TGCAGCACAAGAAAGACCAC-3′ and 5′-CCCTCTCGCTTCAGATGAAC-3′ Tbx, 5′-TTCCCCCTTCGTGTGTTTAG-3′ and 5′-GGACTTCCGTTGTTTCCA-3′ Foxa2, 5′-GACATACCGACGCAGCTACA-3′ and 5′-GGCACTGGAAAGCAGTC-3′ Gata4, 5′-GAGTGTGTCAATTGTGGGGC-3′ and 5′-CTGCTGTGCCCATAGTGAGA-3′ Gata6, 5′-CGGAGGAAAGTACAGAC-3′ and 5′-GAGTAGGGAAAGCGTGCAG-3′ Sox17, 5′-CTCGGGGATGTAAAGGTGAA-3′ and 5′-GCTTCTCTGCCAAGGTCAAC-3′ and Sox7, 5′-GCAGGAAGAAACAAGGCAAG-3′ and 5′-GTGGAGGGGACTAGGTGTGA-3′. PCR products were analyzed by 1.5% agarose gel electrophoresis. Relative levels of mRNA were normalized to the values of Gapdh mRNA for each reaction.
4
Polymer-based Plasma EV Extraction
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EVs were precipitated and extracted using a polymer-based procedure [21 (link)] using 500 µL aliquots of EDTA plasma samples and the ExoQuick-TCTM Exosome Precipitation Solution (System Biosciences, Palo Alto, USA) as recommended by the manufacturer. After resuspension of the pellet, remaining polymers were removed by filtering samples using Cytiva PD SpinTrapTM G-25 (Merck KgaA, Darmstadt, Germany) (see procedural instructions, supplemental digital content S1, extraction of extracellular vesicles, for details of the experiment protocol).
5
Exosome Isolation and Characterization
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Exosomes were isolated with ExoQuick-TCTM Exosome Precipitation Solution (System Biosciences). In brief, culture medium was collected via centrifugation at 3,000 g for 15 min to remove cells and cell debris. Next, 50 mL supernatant was transferred and mixed with 10 mL ExoQuick-TC Exosome Precipitation Solution. After overnight (16 h) incubation, exosomes were collected via centrifugation at 1,500 g for 60 min and subjected to RNA or protein extraction.
For analysis of exosome enrichment, suspended exosomes were incubated with CD63 antibody-conjugated Dynabeads (Thermo Fisher Scientific); CD63 is often used as a marker for exosomes. After magnet-based enrichment reactions, bead-bound exosomes were subjected to CD63-PE antibody staining, followed by washing steps with PBS. Flow cytometry analysis was performed using CellQuest software (BD Biosciences).
For analysis of exosome enrichment, suspended exosomes were incubated with CD63 antibody-conjugated Dynabeads (Thermo Fisher Scientific); CD63 is often used as a marker for exosomes. After magnet-based enrichment reactions, bead-bound exosomes were subjected to CD63-PE antibody staining, followed by washing steps with PBS. Flow cytometry analysis was performed using CellQuest software (BD Biosciences).
6
Exosome Isolation from HepG2 Cells
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HepG2 cells were cultured in 25cm2 flasks. When the confluency reached 60-70%, the cells were washed by PBS twice and counted, and the medium was changed to fresh serum-free DMEM with or without TAPI-2 (Sigma) at the final concentration of 25 μmol/L 18 (link). After 24h, the culture supernatants were collected and concentrated by ultrafilters (Millipore) with molecular weight cut-offs of 10,000. The exosomes were prepared from the concentrated supernatants using the ExoQuick-TCTM Exosome Precipitation Solution (System Biosciences, USA) as described by the manufacturer 7 (link). Meanwhile, the cells were scraped and lysed by cold lysis buffer (Thermo Fisher) with protease inhibitor cocktail (Sigma).
7
Isolation and Characterization of ADSC-Derived Exosomes
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ADSCs were trypsinized and seeded at 5 × 105 cells in a 10-cm dish. After 24 h, the culture medium was collected and centrifuged at 3,000 g for 15 min to remove cells and cell debris. ADSC-Exo were isolated from 10 mL culture media using 2 mL ExoQuick-TCTM Exosome Precipitation Solution per the manufacturer’s instructions (System Biosciences, CA, United States). After overnight incubation at 4°C, the mixture was centrifuged at 10,000 g for 30 min at 4°C. The pellet was washed with 1 mL PBS and centrifuged at 4°C for 15 min at 10,000 g. Then, the purified ADSC-Exos were completely suspended in 50 μL PBS and stored at −80°C for further study. The protein content of the exosomes was quantified using the Bradford method. The purified ADSC-Exo was characterized using the biomarker CD63 and CD9 by Western blot. The morphology and size of ADSC-Exo were observed under a transmission electron microscope (Hitachi, Tokyo, Japan). Nanoparticle tracking analysis (NTA) was used to evaluate the concentration and size distribution of ADSC-Exo. The detailed methods and results for characterization of ADSC-Exo were described in the Supplementary Data . Cardiac and cellular uptake of ADSC-Exo were examined using the PKH-26 labeling kit (Sigma, MO, United States) and observed under a confocal microscope.
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