The MSCExo-Ce solution underwent Cy5 staining for 60 min at 37 °C. Unbound dye was subsequently removed utilizing Exosome Spin Columns (Thermo Fisher). HCECs were exposed to MSCExo-Ce for 12 h, after which they were fixed and imaged.
Exosome spin column
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
The Exosome Spin Columns are a tool used to isolate and purify exosomes from biological samples. They utilize a column-based method to selectively capture and concentrate exosomes, allowing for further downstream analysis or applications.
Automatically generated - may contain errors
Lab products found in correlation
Bodipy tr ceramide, by Thermo Fisher Scientific (7 mentions)
Pkh26, by Merck Group (4 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (3 mentions)
Lsm 880, by Zeiss (3 mentions)
Pkh67 green fluorescent cell linker kit, by Merck Group (3 mentions)
Rnaimax, by Thermo Fisher Scientific (3 mentions)
Pkh26, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Total exosome isolation reagent from cell culture media, by Thermo Fisher Scientific (2 mentions)
Mini67 1kt, by Merck Group (2 mentions)
Facscan lsrii, by BD (2 mentions)
Diluent c, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Digital sight camera ds qi2mc, by Nikon (2 mentions)
Eclipse ti e inverted microscope, by Nikon (2 mentions)
71 protocols using exosome spin column
1
Fluorescent Labeling of Extracellular Vesicles
2
Exosome Isolation and Characterization
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Lipofectamine 2000, Dulbecco's modified Eagle's medium (DMEM), α‐minimal essential medium (α‐MEM), Opti‐MEM, fetal bovine serum, horse serum, penicillin‐streptomycin, 0.25% trypsin‐EDTA, DiO dye, Exosome Spin Columns, Micro BCA Protein Assay kit, as well as CD11b, CD86, CD206, CD11c, CD80 monoclonal antibodies were purchased from Thermo Fisher Scientific, USA. Calnexin, CD9, CD63 and CD81, FASL, Caspase‐3, Caspase‐8, and PARP antibodies were purchased from Abcam. 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), RNAlater, dimethyl sulfoxide (DMSO), LysoTracker Green DND‐26, isothiocyanate‐labeled phalloidin and Hoechst 33342 were purchased from Sigma Aldrich, USA. Filter membranes and ultrafilter tubes were purchased from Millipore (Shanghai, China). Cy5‐labeled siRNA (Cy5‐NC), siPLK1 (targeting PLK1), and siPD‐L1 (targeting PD‐L1) were supplied by Suzhou Ribo Life Science Co., Ltd. (Suzhou, China) or Suzhou Biosyntech Co., Ltd (Suzhou, China). All the primers were provided by BioSune Co., Ltd (Shanghai, China). DCFH‐DA, Annexin V‐FITC/PI Apoptosis Detection Kit, and Hieff qPCR SYBR Green Master Mix were purchased from Yeasen (Shanghai, China). Chlorin e6 (Ce6) was purchased from Macklin Biochemical Technology Co., Ltd (Shanghai, China). Optimal cutting temperature (OCT) compound was from Sakura Finetek USA, Inc. (Torracne, CA90501, USA).
3
Exosome Uptake Visualization Protocol
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1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil; Beyotime, Shanghai, China) was used to indicate the endocytosis of exosomes by NK92-MI cells through staining of the cytoplasm and intracellular membrane. Briefly, the SW480-exosome suspension was incubated with Dil (1:2000, Sigma) and washed through Exosome Spin Columns (MW3000, Life, Thermo Fisher, USA) to obtain Dil-labeled SW480 exosomes, which were incubated with NK92-MI cells. The chamber cells were fixed with 4% paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime). Finally, the endocytosis of the recipient cells was examined under a fluorescence microscope (Nikon ECLIPSE C1) equipped with a DS-U3 imaging system (Nikon).
4
Isolation and Characterization of Extracellular Vesicles
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EVs were isolated from PL‐based preparations2 , 46 , 47 , 48 , 49 , 51 by sequentially ultracentrifugation (500 rcf for 10 min; 2000 rcf for 10 min; 100,000 rcf for 1 h). EV pellet was then resuspended to reconstitute the initial PL volume and subsequently used at 5%, 10% or 20% in the medium for the experiments. For FACS analysis and uptake assay in human umbilical vein endothelial cell (HUVEC), 100 μl of EVs were stained for 10 min at 37°C with 5 μM 5(6)‐CFDA‐SE [5‐(and‐6)‐carboxyfluorescein diacetate, succinimidyl ester] (CFSE; Invitrogen/Thermo Fischer Scientific) according to the manufacturer's instructions. Excess dye was removed using Exosome Spin Columns (Thermo Fischer Scientific) following the manufacturer's recommendations.
5
Isolation and Labeling of Exosomes from HeLa and HT1080 Cells
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The HeLa and HT1080 cell lines were purchased from Sigma-Aldrich (ECACC, 93021013 and 85111505). Cells were cultured until 80% confluency and washed 3 times with DMEM. Then, the medium was switched to DMEM. They were then conditioned for 48 hours, after which exosomes were isolated as previously described11,12. In brief, exosomes were isolated by ultrafiltration. Conditioned medium was filtered through 0.22 µm Steriflip filters to remove cellular debris and large vesicles. The filtrate was then added to Amicon Ultra-15 100 kDa filters (Millipore, SCGP00525) to centrifuge at 5,000 × g for 5 min. The flow-through was discarded and the concentrated exosomes were collected and washed with PBS three times before being stored at -80°C.
Exosome labeling was performed using 10 uM DiI or DiO (Thermo Fisher, V22889), incubated for 20 minutes at 4°C. Then, Exosome Spin Columns (Thermo Fisher, 4484449) were used to remove the unincorporated dye.
Exosome labeling was performed using 10 uM DiI or DiO (Thermo Fisher, V22889), incubated for 20 minutes at 4°C. Then, Exosome Spin Columns (Thermo Fisher, 4484449) were used to remove the unincorporated dye.
6
In vivo Imaging of Fluorescently Labeled Extracellular Vesicles
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In vivo imaging experiments were performed as previously described [71 (link)]. EV fraction (15 µg) was incubated with 5 µM Cy7 Mono NHS Ester (GE Healthcare, Buckinghamshire, UK) for 90 min at 37 °C. The unincorporated dye was removed using Exosome Spin Columns (MW. 3000) (ThermoFisher). Animal experiments were performed in compliance with the guidelines of the Ethics Committee of Animal Care and Experimentation of Tokushima University (approval number T29-31). Eight-week-old female BALB/c mice (CLEA, Tokyo, Japan) were administered with 15 µg Cy7-labeled EVs per mouse by intraperitoneal injection. At 30 min after the injection, Cy7 fluorescence in the various organs of mice was analyzed by the IVIS Spectrum imaging system (Caliper Life Sciences, A PerkinElmer Company, Hopkinton, MA, USA).
7
Isolation of Extracellular Vesicles from BMDM
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For isolation of extracellular vesicles, the Total Exosome Isolation Reagent from cell culture media (Thermo Scientific) was used. After differentiation, BMDMs were seeded in a density of 1 × 107 cells/10 ml dish. FCS supplement in BMDM media was replaced by exosome‐depleted FCS (Thermo Scientific). In order to avoid carryover of LPS after the LPS priming step, BMDMs were rinsed twice with pre‐warmed PBS before stimulation with ATP. After ATP stimulation, cell culture media was harvested and centrifuged at 2,000 ×g for 30 min at 4°C to remove cells and debris. The supernatant was transferred into a new tube and mixed with the reagent mixture well by vortexing. Samples were incubated overnight at 4°C. After incubation, samples were centrifuged at 10,000 ×g for 1 h at 4°C. The supernatant was carefully discarded. Extracellular vesicles were resuspended in PBS. To remove ATP and possible contaminants, Exosome Spin Columns (MW3000, Thermo Scientific) were used according to the manufacturer's protocol. The protein content of the EVs was determined using BCA protein assay (Pierce), and subsequent stimulations and injections were carried out using equal amounts of EV protein.
8
Fluorescent Labeling of Extracellular Vesicles
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EVs were labelled with lipophilic red fluorescent dye (PKH26, Sigma-Aldrich), according to the manufacturer’s protocol. Briefly, 0.32 μl PKH26 was mixed with 5 μg EV samples in 40 μl diluent C, and incubated for 5 min at room temperature. dfPBS was used as a negative control. The labelling reaction was stopped by adding 50 μl chilled dfPBS, and subjected to Exosome Spin Columns (MW 3000, ThermoFisher) at 750g for 2 min to remove the free dye and enrich the labelled EVs, which were adjusted to 5 μg/100 μl for the neuronal EV uptake assay.
9
Labeling and Visualization of Extracellular Vesicles
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As described [8 (link),45 (link),46 ], 20 μg of EV fraction was incubated with 10 μM BODIPY TR Ceramide (ThermoFisher) for 20 min at 37°C. Excessive dye was removed using Exosome Spin Columns (MW. 3000) (ThermoFisher). The labelled EVs were added to the culture medium of HSC-3 cells in 96-well plates at 25 μg/mL. Cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.5% Tween-20 in PBS for 5 min. Cells were incubated with ActinGreen488 (ThermoFisher) for 30 min and with DAPI for 5 min. Fluorescence images of random four fields were taken using a Floid® Imaging Station (ThermoFisher) and fluorescence-positive cells in each field were counted.
10
EV Uptake in Cultured Fibroblasts
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To track EV uptake by cultured fibroblasts, purified EV were labeled with PKH26, a red membrane dye (Sigma-Aldrich), according to the manufacturer's protocol. Briefly, 300 μl of EV was suspended into 100 μl of Diluent C, which was mixed with 1.4 μl of PKH26 dye. The labeling reaction was stopped by adding an equal volume of EV-free FBS. Exosome Spin Columns (Cat. 4484449, Thermo Fisher Scientific) were used to remove unincorporated PKH26. The cultured fibroblasts in the slide chamber were incubated with labeled EV at 37°C for 24 h. After incubation, cells were stained with Calcein AM (5 μM). Cells were fixed with 2% formaldehyde for 5 min and mounted with DAPI containing ProLong Gold Antifade medium (Thermo Fisher Scientific). Images were taken with an FV1000 confocal microscope (Olympus, Japan).
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