Conditioned media were obtained from E0771/CD63-RFP cells grown at subconfluence for three to four days in exosome-depleted growth media (Gibco Laboratories, Carlsbad, CA, USA). TEXs were isolated from collected conditioned media by using the Exo-spin™ Exosome Purification Kit (Cell Guidance Systems, Cambridge, UK). The size distribution and concentration of exosomes were determined using a NanoSight NS500 (Malvern, Grovewood Road, UK) equipped with a 642-nm laser and a charge-coupled device (CCD) camera, and data were analyzed using Nanoparticle Tracking Analysis software [23 (link)].
Exo spin exosome purification kit
Manufactured by Cell Guidance Systems
Sourced in United Kingdom, United States
The Exo-spin™ Exosome Purification Kit is a laboratory equipment product designed for the isolation and purification of exosomes from various biological samples, such as cell culture media or bodily fluids. The kit utilizes a proprietary spin column-based method to efficiently capture and concentrate exosomes, allowing for downstream analysis and characterization.
Automatically generated - may contain errors
Lab products found in correlation
Ns500, by Malvern Panalytical (1 mentions)
Exosome depleted fbs, by Thermo Fisher Scientific (1 mentions)
Cyquant ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Capricorn (1 mentions)
Pegylated liposomal doxorubicin, by Johnson & Johnson (1 mentions)
Em 910, by Zeiss (1 mentions)
Zetaview instrument, by Particle Metrix (1 mentions)
Optima max xp ultracentrifuge, by Beckman Coulter (1 mentions)
Qev column, by Izon Science (1 mentions)
Protease inhibitor, by Roche (1 mentions)
9 protocols using exo spin exosome purification kit
1
Isolation and Characterization of Tumor-Derived Exosomes
2
Exosome Isolation from Conditioned Media
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PBP+SP-EVs were isolated from CCM using the Exo-spin™ Exosome Purification Kit (Cell Guidance Systems, Cambridge, UK) according to the manufacturer’s instructions. Briefly, CCM was mixed with Buffer A (2:1, v/v) and incubated overnight at 4°C on a roller mixer. Afterwards, the samples were centrifuged at 20,000 g for 60 mins at 4°C. The supernatant was discarded and the pellet was resuspended in 100 μl of PBS. The resuspension was loaded into a spin-column pre-washed with PBS. The loaded column was centrifuged at 50 g for 60 s. The fraction containing the PBP+SP-EVs was then eluted with 200 μl of PBS by low speed centrifugation for 1 min.
3
Isolation and Characterization of Exosomes from db/db Mice
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ASCs and DFbs of db/db mice were isolated as previously described. ASCs or DFbs were cultured in exosome-free Dulbecco’s modified Eagle’s medium (DMEM; Gibco Invitrogen/Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen/Life Technologies, Carlsbad, CA, USA) at 37 °C in a 5% CO2 incubator. Culture supernatants were collected from conditioned mediums, including 5% exosome depleted FBS (Thermo Fisher Scientific, Waltham, MA, USA) after 3 days. The supernatants were first centrifuged at 300× g for 10 min to remove all cells and any cell debris. The exosome fraction was precipitated from the supernatant with reagents from the Exo-spin exosome purification kit (Cell Guidance Systems, St. Louis, MO, USA). Purification of the exosomes was then performed with centrifuges with the microcentrifuge collection tube where the final elute of purified exosomes was obtained. The morphological analysis of collected exosomes were observed by transmission electron microscopy (Hitachi-7000FA, Tokyo, Japan). The protein expression of exosome-associated general markers including CD9, CD63, and CD81 (Abcam) were analyzed by Western blotting and flow cytometry. The particle size and distribution of db/db mice ASC-Exo were identified by NanoSight LM10 (NanoSight, Amesbury, UK).
4
Exosome Purification and Characterization
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PEGylated liposomal doxorubicin was obtained from Janssen (Belgium). Exo-spin™ exosome purification kit was obtained from Cell Guidance systems (UK). CyQUANT™ LDH cytotoxicity assay kit and 3.9-μm latex beads were purchased from Invitrogen (USA). Exosome-free fetal bovine serum (FBS) was from System Biosciences (USA) and RPMI 1640 was from Capricorn (Germany). BCA protein assay kit was purchased from Parstous Biotechnology (Iran). Anti-CD63/PE antibody and Isotype control mouse IgG/PE was from BioLegend (USA). Annexin V-FITC/PI kit was obtained from MabTag (Germany).
5
Plasma Exosome Isolation Protocol
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Blood was drawn into ethylenediaminetetraacetic acid-containing tubes (5 ml). Plasma was isolated using centrifugation at 300 g for 10 min and further spun down at 2000 g for 20 min to remove dead cells and cell debris. Then, plasma was harvested, immediately aliquoted, and banked in −80°C until future use.
Plasma was centrifuged for 20 min at 2000 ×g, 4°C. The resulting supernatant was further centrifuged at 16000 ×g, 4°C for 30 min. The supernatant was filtered through a 0.22-μm filter. Then, exosomes were isolated using the ExoSpin Exosome Purification Kit (Cell Guidance Systems, USA) according to the manufacturer's instructions. Finally, the exosomes were stored at −80°C.
Plasma was centrifuged for 20 min at 2000 ×g, 4°C. The resulting supernatant was further centrifuged at 16000 ×g, 4°C for 30 min. The supernatant was filtered through a 0.22-μm filter. Then, exosomes were isolated using the ExoSpin Exosome Purification Kit (Cell Guidance Systems, USA) according to the manufacturer's instructions. Finally, the exosomes were stored at −80°C.
6
Exosome Isolation and Characterization from NK Cells
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To obtain NKExos, NK92MI cells were cultured in medium containing 10% exosome-depleted FBS (Biowest, Nuaillé, France) for 72 h. Cells and cellular debris were removed by centrifugation at 300× g for 10 min and at 16,000× g for 30 min at 4 °C. Next, exosomes were concentrated using 100K Amicon® Ultra-15 centrifugal filters (Merck Millipore, Co cork, Ireland) and purified using Exo-spin™ Exosome Purification kit (Cell Guidance Systems, Cambridge, UK) according to manufacturer’s instructions. Purified exosomes were quantified using the CD63 Trific™ Exosome detection kit (Cell Guidance Systems) according to manufacturer’s instructions and stored at 80 °C until further use. Immunogold staining and Transmission Electron Microscopy (TEM) was performed by the DKFZ Core Facility Unit Electron Microscopy (Heidelberg, Germany). For immunostaining, CD56 (clone: MEM-188, Biolegend), CD63 (clone: H5C6, BD Biosciences), and isotype controls were used followed by a rabbit-anti-mouse antibody conjugated to 5 nm gold particles. The samples were analyzed using an EM910 (Zeiss, Oberkochen, Germany) transmission electron microscope at 63,000× magnification. Particle size of exosomes was determined by Nanoparticle Tracking Analysis (NTA) using a ZetaView® Instrument (Particle Metrix, Inning am Ammersee, Germany) by Cell Guidance Systems.
7
Exosome Isolation from Breast Cancer Cells
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A stable MDA-MB-231/CD63-RFP cell line overexpressing the exosomal CD63-RFP fusion protein was generated for monitoring RFP-tagged exosomes. Conditioned media were obtained from MDA-MB-231/CD63-RFP grown at sub-confluence for 3 to 4 days in growth media containing serum depleted of bovine exosomes (Gibco Laboratories, Carlsbad, CA, USA). For isolation of exosomes from conditioned media, the Exo-spin Exosome Purification kit was used according to the manufacturer’s instructions (Cell Guidance Systems, Cambridge, UK). Purified exosomes were then stored at −80°C until use.
8
Isolation of Small Extracellular Vesicles
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Small EVs from conditioned cell-culture media (30 mL per EV sample) were separated using the Exo-spin exosome purification kit (Cell Guidance Systems, Cambridge, UK), a combination of EV precipitation followed by size exclusion chromatography, according to the manufacturer's instructions. Small EVs from 0.5 mL serum samples were isolated as previously described. 14 In short, we used size exclusion chromatography using the sepharose-based qEV columns (iZON Science, Christchurch, New Zealand). The small EVs were eluted with Hank's balanced salt solution (HBSS). Fractions 8 to 10 of 500 μL each were collected and pooled and a protease inhibitor (Roche, Basel, Switzerland) was added. Subsequently, 1.5 mL of this mixture were concentrated to a final volume of 120 μL by ultracentrifugation (1 hour and 45 minutes, 110 000 g, 4 C) using the TLA-55 rotor and the Optima MAX-XP ultracentrifuge (both Beckman Coulter, Brea, California).
9
Exosome Isolation from Cell Culture
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For EV isolation, cells were grown in EV-depleted medium, which was generated by ultracentrifugation of fetal bovine serum at 110,000g for 16 hours at 4 C. EVs were isolated using Exo-spin Exosome Purification Kit (Cell Guidance Systems, Cambridge, UK) according to manufacturer's protocol, with the addition of a filtration step with 0.22-mm filters after the final preclearing centrifugation.
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