Exoquick tc exosome isolation kit

Manufactured by System Biosciences

Sourced in United States

The ExoQuick-TC exosome isolation kit is a laboratory tool designed to isolate extracellular vesicles, including exosomes, from cell culture media and other liquid samples. The kit utilizes a proprietary reagent to facilitate the precipitation and recovery of these vesicles from the sample.

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Lab products found in correlation

Bca protein assay kit, by Beyotime (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Cytomics fc500, by Beckman Coulter (1 mentions) Gw4869, by Merck Group (1 mentions) Anti bcl 2, by Cell Signaling Technology (1 mentions) Exosome depleted fetal bovine serum, by System Biosciences (1 mentions) Cisplatin, by Qilu Pharmaceutical (1 mentions) Cell counting kit 8 cck 8, by BestBio (1 mentions) Thp 1 monocytes, by American Type Culture Collection (1 mentions) Exoquick exosome precipitation solution, by System Biosciences (1 mentions) Tecnai f20, by Thermo Fisher Scientific (1 mentions) Bca assay, by Takara Bio (1 mentions) Tem 2000 ex 2, by JEOL (1 mentions) Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions) Nanodrop 3300, by Thermo Fisher Scientific (1 mentions) Nano zs90, by Malvern Panalytical (1 mentions) 17β estradiol, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Insulin, by Merck Group (1 mentions) Triiodothyronine, by Merck Group (1 mentions)

Close spelling variants of this product

ExoQuick-TC (231 citations) ExoQuick (195 citations) ExoQuick-TC kit (67 citations) ExoQuick kit (64 citations) ExoQuick-TCTM (12 citations) Exosome isolation kit (9 citations) ExoQuick exosome isolation kit (7 citations) ExoQuick isolation kit (4 citations) ExoQuick-TC isolation kit (3 citations)

10 protocols using exoquick tc exosome isolation kit

NSCLC Cell Line Experimental Protocol

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The human NSCLC cell lines PC9 and A549 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were maintained in RPMI 1640 medium (Gibco, USA) and supplemented with 10% exosome-depleted fetal bovine serum (System Biosciences, USA).
Gefitinib (Santa Cruz, USA) and cisplatin (Qilu pharmaceutical, China) were re-suspended in dimethyl sulfoxide (DMSO) and phosphate buffer solution (PBS), respectively, and stocked at a concentration of 10 mM at −20°C. The anti-LC3 and anti-P62 antibodies as well as GW4869 were purchased from Sigma (Sigma-Aldrich, USA). Anti-Bcl-2 and anti-Bax antibodies were purchased from Cell Signaling Technology (CST, USA). An Exoquick-TC^™ exosome isolation kit and anti-CD63 antibody were purchased from SBI (System Bioscience, USA). Anti-β−actin and anti-rabbit and anti-mouse IgG peroxidase-conjugated secondary antibodies were obtained from Bioworld (USA). A cell counting kit-8 (CCK-8) was purchased from Bestbio (Bestbio, China). A BCA protein assay kit was purchased from Beyotime (Beyotime, China). Cell apoptosis was measured using an Annexin V-FITC/propidium iodide (Bestbio, China) double-staining assay and flow cytometry (Cytomics^™ FC 500, Beckman Coulter, USA).

Li X.Q., Liu J.T., Fan L.L., Liu Y., Cheng L., Wang F., Yu H.Q., Gao J., Wei W., Wang H, & Sun G.P. (2016). Exosomes derived from gefitinib-treated EGFR-mutant lung cancer cells alter cisplatin sensitivity via up-regulating autophagy. Oncotarget, 7(17), 24585-24595.

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Exosome Extraction from Cell Culture

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When the cells were 70-80% confluent, the culture medium was replaced with serum-free DMEM for 48 h. Then, exosomes were extracted from the medium using an ExoQuick-TC exosome isolation kit (SBI, Palo Alto, CA, USA) in accordance with the manufacturer’s protocol. In detail, the serum-free DMEM was collected and centrifuged for cell debris removal. Then, the supernatant was transferred to a sterile vessel, mixed with precipitation solution and incubated at 4° C overnight. On the second day, the mixture was centrifuged, the supernatant was discarded and the exosome pellet was isolated. The exosomes were stored in a -80° C freezer or used immediately for experiments.

Wu J., Fang X., Huang H., Huang W., Wang L, & Xia X. (2021). Construction and topological analysis of an endometriosis-related exosomal circRNA-miRNA-mRNA regulatory network. Aging (Albany NY), 13(9), 12607-12630.

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Isolation and Characterization of Exosomes

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Cells lines for ES cells (CGR8, mouse embryonic stem cells) and mouse embryonic fibroblasts (MEF) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). ES cells were cultured in a gelatin-coated tissue culture plate in DMEM containing 15% ES-fetal bovine serum (ES-FBS), β-mercaptoethanol, glutamine, penicillin/streptomycin (P/S), leukemia inhibitory factor (LIF), and sodium pyruvate (NaP). MEF cells were cultured in DMEM containing 10% FBS, NaP, P/S, and glutamine. After 48 h, the cell culture medium was discarded and serum-free knockout DMEM and P/S were added (ThermoFisher Scientific). Exosome (Exos) isolation was performed according to the manufacturer’s protocol of the Exoquick TC exosome isolation kit (SBI, Palo Alto, CA, USA). The isolated Exos were characterized by performing western blot on exosome markers HSP-70 and CD63, as we have recently shown [22 (link)].

Dessouki F.B., Kukreja R.C, & Singla D.K. (2020). Stem Cell-Derived Exosomes Ameliorate Doxorubicin-Induced Muscle Toxicity through Counteracting Pyroptosis. Pharmaceuticals, 13(12), 450.

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Exosome Isolation and Inflammation Induction

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Sol-8 cells (a mouse muscle cell line) were purchased from ATCC, USA and cultured in DM (Differentiation Medium) containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), glutamine, sodium pyruvate and penicillin-streptomycin (P/S). To induce exogenous inflammation, we prepared conditioned medium (CM) from THP-1 monocytes D r a f t obtained from ATCC, USA. THP-1 cells were grown in suspension medium for 48 hours, centrifuged, supernatant filtered (using 0.22 µl filter, Millipore) and saved as THP-1 conditioned medium (THP-1 CM).
ES cells (CGR8, a mouse embryonic cell line) and mouse embryonic fibroblasts (MEF) were cultured as reported previously (Singla and McDonald, 2007; Lee et al., 2007) . For isolation of exosomes, cell culture medium was replaced with serum free knockout DMEM supplemented with P/S and glutamine. ES and MEF exosomes were prepared using ExoQuick TC exosome isolation kit (SBI, USA) as per manufacturer's instructions and appropriate modifications were used as necessary. We used GW4869 compound which served as an exosome inhibitor.

Tavakoli Dargani Z., Singla R., Johnson T., Kukreja R, & Singla D.K. (2018). Exosomes derived from embryonic stem cells inhibit doxorubicin and inflammation-induced pyroptosis in muscle cells. Canadian journal of physiology and pharmacology, 96(3).

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Exosome Isolation and Characterization

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Exosomes were isolated from cell culture supernatants by using ExoQuick-TC Exosome Isolation kit (System Biosciences, Palo Alto, USA) [19 (link)] and from serum by using ExoQuick exosome precipitation solution (System Biosciences, Palo Alto, USA) [20 (link)] as described previously, respectively. Briefly, the culture supernatant was collected and centrifuged at 4000 × g for 30 min to remove cell debris, and one-fifth of ExoQuick-TC was added to supernatant following by incubating overnight at 4 °C. Exosomes were isolated by centrifugation at 1500 × g for 30 min. Serum was harvested and centrifuged at 500 × g and 10,000 × g for 10 min to remove intact cells and cell debris, individually. 1 mL serum was mixed with 250 μL of solution and incubated for 30 min at 4 °C, and then centrifuged at 1500 × g for 30 min to isolate exosomes. Each pellet was resuspended with PBS and the protein concentration of exosome was determined by BCA assay (TAKARA, Japan). The characterization of exosomes was analyzed by transmission electron microscopy (TEM) (FEI, Tecnai F20). The density and size of exosomes were tracked by nanosight particle tracking analysis (NTA) (NS300 system, NanoSight technology, Malver, UK).

Zhang C., Wang X.Y., Zhang P., He T.C., Han J.H., Zhang R., Lin J., Fan J., Lu L., Zhu W.W., Jia H.L., Zhang J.B, & Chen J.H. (2022). Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts. Cell Death & Disease, 13(1), 57.

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Exosome Isolation and Depletion from BM-MSC Secretome

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Exosomes (Exo) were isolated from the concentrated BM-MSC CM using ExoQuick-TC exosome isolation kit (System Biosciences) (Figure S1B). Transmission electron microscope (TEM) was used to evaluate Exo morphology and Nanosight analysis was employed to determine size distribution of Exo.
GW4869 (an exosome release inhibitor, 10 μM) was utilized to treat BM-MSCs [15 , 18 (link)]. After 72hr treatment, the BM-MSC CM was collected, centrifuged, and concentrated to obtain exosome-depleted MSC-CM (Figure S1C).

Scott S.R., March K.L., Wang I.W., Singh K., Liu J., Turrentine M., Sen C.K, & Wang M. (2021). Bone marrow- or adipose-mesenchymal stromal cell secretome preserves myocardial transcriptome profile and ameliorates cardiac damage following ex vivo cold storage. Journal of molecular and cellular cardiology, 164, 1-12.

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Exosome Isolation and Characterization from CAFs and NFs

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After culturing CAFs and NFs for 72 hr, the supernatants were collected and subjected to gradient centrifugation (300 g, 2000 g and 12000 g, respectively, for 30 minutes) followed by filtering through a 10K Centricon Plus-70 ultrafiltration tube (Millipore, USA) to remove cell debris and impurities. The remaining supernatant was purified by an EXO Quick-TC exosome isolation kit (EXOTC50A, System Biosciences, USA) according to the instructions. The protein concentration of the obtained exosome suspension was determined with a BCA Protein Assay Kit (Biyuntian, China), and the newly prepared exosomes were subjected to transmission electron microscopy (TEM) observation with TEM-2000 EX II (JEOL, Tokyo, Japan) as previously described 37 . The size distribution of exosomes was determined by a Zetasizer Nano ZS90 (Malvern, UK) light scattering size potentiometer according to a previous report 38 (link). Cell proliferation was assessed with a Cell Counting Kit-8 (Dojindo, Japan) according to the user manual with 5 repeats, while absorbance at 450 nm was recorded using a microplate spectrophotometer (NanoDrop 3300, Thermo, US).

Jin Y., Meng Q., Zhang B., Xie C., Chen X., Tian B., Wang J., Shih T.C., Zhang Y., Cao J., Yang Y., Chen S., Guan X., Chen X, & Hong A. (2021). Cancer-associated fibroblasts-derived exosomal miR-3656 promotes the development and progression of esophageal squamous cell carcinoma via the ACAP2/PI3K-AKT signaling pathway. International Journal of Biological Sciences, 17(14), 3689-3701.

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Exosome Isolation from Cell Culture

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The ExoQuick-TC™ exosome isolation kit (System Biosciences; Palo Alto CA, USA) was used according to the manufacturer’s instructions on concentrated culture supernatant. Cells and bigger particles were removed by centrifugation at 3000g for 15 min. The supernatant was then added with 1/6 volumes of ExoQuick-TC™ precipitation reagent. The reagent was mixed with the supernatant and stored at 4 °C overnight. The next day, the sample was centrifuged twice at 1500g for 30 min and 5 min, respectively, to take off the supernatant. The pellet was resuspended in 100 μl PBS, supplemented with 1 mM PMSF.

Keysberg C., Hertel O., Schelletter L., Busche T., Sochart C., Kalinowski J., Hoffrogge R., Otte K, & Noll T. (2021). Exploring the molecular content of CHO exosomes during bioprocessing. Applied Microbiology and Biotechnology, 105(9), 3673-3689.

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Isolation and Characterization of HUVECs

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Human umbilical vein endothelial cells (HUVECs) (C2517A, Lonza, Walkersville, MD, USA) were cultured in endothelial growth medium (EGM-2) purchased from Lonza Bioscience (Walkersville, MD, USA). The HUVECs were maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. Norepinephrine, acetylcholine, triiodothyronine (T3), thyroxine (T4), insulin-like growth factor (IGF), cortisol, aldosterone, insulin, glucagon, 5α-dihydrotestosterone (DHT), 17β-estradiol (E2), progesterone (P4), estrone (E1), estriol (E3), fulvestrant, N(ω)-nitro-L-arginine methyl ester (L-NAME), 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), GW4869, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO, USA). An ExoQuick-Tc exosome isolation kit was purchased from System Biosciences (Palo Alto, CA, USA). The polyclonal antibody against APE1/Ref-1 was obtained from MediRedox, Inc. (Daejeon, Korea) and, the anti-CD63 antibody was obtained from Biobyt (Cambridge, UK). The monoclonal antibody against anti-heat shock protein-70 (HSP70, C92F3A-5) was obtained from Enzo Life Science (Farmingdale, NY, USA), the anti-CD9 (C-4) antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and anti-ALG-2 interacting protein X (Alix, 3A9) antibody was obtained from Thermo-Fisher Scientific Inc (Waltham, MA, USA).

Lee Y.R., Joo H.K., Lee E.O., Kim S., Jin H., Choi Y.H., Kim C.S, & Jeon B.H. (2021). 17β-Estradiol Increases APE1/Ref-1 Secretion in Vascular Endothelial Cells and Ovariectomized Mice: Involvement of Calcium-Dependent Exosome Pathway. Biomedicines, 9(8), 1040.

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Exosome Isolation and Characterization from NPC

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Exosomes were extracted from individual serum samples of NPC patients and conditional media of cultured NPC cells by differential ultracentrifugation using an ExoQuick-TC exosome isolation kit (System Biosciences, Palo Alto, CA). Briefly, the collected serum and conditioned medium samples were sequentially centrifuged at 300 × g for 10 min and 2,000 × g for 10 min and mixed with ExoQuick-TC solution and centrifuged at 10,000 × g for 30 min. The supernatants were filtered using a 0.22 μm filter and centrifuged at 100,000 × g for 70 min (all centrifugation at 4°C). The resulting pellets were re-suspended in PBS.
The collected exosomes were placed on the copper mesh and stained with 2% uranyl acetate, followed by examination under a transmission electron microscope (G2 Spirit; FEI). Brownian motion and concentrations of exosomal samples were analyzed using ZetaView 8.04.02 SP2 (ZetaView PMX 110, Particle Metrix, Germany). Furthermore, exosomes were characterized by western blot using antibodies against HSP70 (1:500; 66183-1-Ig; Proteintech), CD63 (1:500; ab216130; Abcam), and TSG101 (1:500; ab30871; Abcam).
In addition, different groups of cells (1 × 10⁵ cells/well) were treated with 10 μg exosomes for 48 h. Cells were analyzed in subsequent experiments. The purified exosomes were labeled with PKH67 (Sigma-Aldrich, St. Louis, MO).³³ (link)

Yang W., Tan S., Yang L., Chen X., Yang R., Oyang L., Lin J., Xia L., Wu N., Han Y., Tang Y., Su M., Luo X., Yang Y., Huang L., Hu Z., Tao Y., Liu L., Jin Y., Wang H., Liao Q, & Zhou Y. (2022). Exosomal miR-205-5p enhances angiogenesis and nasopharyngeal carcinoma metastasis by targeting desmocollin-2. Molecular Therapy Oncolytics, 24, 612-623.

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