After 48 h, we collected the culture medium from the bottom wells in order to determine the amount of exosomes that could have passed through the membrane filter during the incubation period. The media collected from the bottom wells (6 ml of media total, from 3 identical wells) with or without addition of exosomes were collected and subjected to exosome isolation [30 (link)]. Briefly, the media were transferred to 15 ml Beckman tubes (Beckman Coulter, Indianapolis, IN; catalog number #342082) and centrifuged at 2000 g for 30 min to sediment contaminating cells. Clean cell-free media (6 ml each) were transferred into fresh 15 ml centrifuge tubes and 3 ml of exosome isolation reagent was added (Invitrogen, Carlsbad, CA; catalog number 4478359) and mixed by inverting the tubes three times. Exosomes were precipitated by incubating the mix overnight at 4 °C and collected by centrifuge at 10,000 g for 1 h at 4 °C. Supernatant was removed by aspiration and the exosome pellet was suspended in 100 μl of PBS and stored at −80 °C until use.
Exosome isolation reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Exosome Isolation Reagent is a product designed to facilitate the isolation and purification of exosomes from various biological samples, such as cell culture media or bodily fluids. The reagent utilizes a proprietary precipitation-based method to selectively precipitate and concentrate exosomal particles, allowing for their subsequent collection and analysis.
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Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Beckman tube, by Beckman Coulter (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Ripa solution, by Solarbio (1 mentions)
Alexa fluor 568 nhs ester, by Thermo Fisher Scientific (1 mentions)
8 chamber slide, by Thermo Fisher Scientific (1 mentions)
0.22 μm syringe filter, by Merck Group (1 mentions)
Ti45 rotor, by Beckman Coulter (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Steriflip, by Merck Group (1 mentions)
Exosome depleted fbs, by Thermo Fisher Scientific (1 mentions)
0.22 mm filter, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Pkh26 fluorescent labeling kit, by Merck Group (1 mentions)
Bmscs, by ScienCell (1 mentions)
Ivis spectrum imaging system, by PerkinElmer (1 mentions)
27 protocols using exosome isolation reagent
1
Exosome Isolation from Cell Culture
2
Exosome Isolation from BMSCs
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BMSCs at passages three to six were cultured in exosome-depleted FBS for 72 h.
The conditioned medium was collected, and Exosome Isolation Reagent (Invitrogen, USA) was used in accordance with the manufacturer’s protocol to isolate exosomes.
The conditioned medium was collected, and Exosome Isolation Reagent (Invitrogen, USA) was used in accordance with the manufacturer’s protocol to isolate exosomes.
3
Isolation and Characterization of BMSC-Derived Exosomes
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The exosomes were isolated from the culture medium of BMSCs using an exosome isolation reagent (Cat#: 4478359, Invitrogen). Briefly, the media of BMSCs were centrifuged at 2,000 × g for half an hour to harvest the supernatant. Then, the Total exosome isolation reagents were appended into the supernatant and vortexed for the sufficient mixture. The mixture was hatched at 4°C for 24 h and centrifuged at 10,000 × g for 60 min to collect the pellet. The pellet was re-suspended into phosphate buffer saline (PBS, Cat#: P1020, Solarbio, Beijing, China) to obtain the isolated BMSC-derived exosomes (BMSC-Exo). The BMSC-Exo was imaged under transmission electron microscopy (TEM) (Cat#: 1200EX, Jeol, Japan). Besides, the exosomal markers, containing CD9, CD63, and calnexin, were examined via western blot after the treatment of the RIPA solution (Cat#: R0010, Solarbio).
4
Leishmania Exosome Isolation Protocol
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Viable promastigotes of L. shawi and L. guyanensis kept in Schneider’s medium were centrifuged at 1800× g. A fresh medium supplemented with exo-free FBS was added, and parasites were incubated at 26 °C for 24 h. Cultures were again centrifuged at 1800× g for 10 min, and the pellet was transferred to the fresh medium and incubated at 26 °C for 72 h. After this period, the cultures were centrifuged at 1800× g for 10 min to remove the parasites, and supernatants were further centrifugated at 2000× g for 30 min to remove cellular debris. The supernatant was collected, and the exosome isolation reagent (Invitrogen, Carlsbad, CA, USA) was added at a ratio of 1:2, according to the manufacturer’s instructions, and the supernatant was incubated for 24 h at 4 °C. After incubation, the EV solution was centrifuged at 10,000× g for 1 h at 4 °C. Pellets (rich in EVs) were resuspended in 1× phosphate-buffered saline (PBS) and used immediately or stored at −80 °C for further assays. In parallel, sterile Schneider’s medium supplemented with 10% exo-free FBS followed the same protocol of EV isolation, and the obtained solution was used as a negative control of the EV isolation method (ImC). The proteins in the final EV solution were quantified in the NanoDrop 1000® spectrophotometer (Thermo Scientific, Waltham, MA, USA).
5
Isolation and Characterization of AR42J Exosomes
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Culture medium was collected when AR42J cells reached 70%–80% confluency in the culture dish. The collected culture medium was centrifuged at 2000 × g with a centrifugation radius of 11 cm for 30 min, thereby removing cell debris and apoptotic bodies. The supernatant was retained and 0.5 volume of exosome isolation reagent (Invitrogen, CA) was added, and the samples were incubated at 4 ℃ overnight. The samples were centrifuged at 10000 × g and 4 ℃ at with a radius of 11 cm for 60 min, and the supernatant was discarded. The samples were resuspended in PBS and stored in separate package at –80 ℃. According to the operation instructions, the particle size distribution and concentration of AR42J exosomes were measured using a Zeta View instrument.
6
Alexa Fluor 568 Labeling of Teratocytes
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For Alexa-NHS labeling, teratocytes were collected and cultured with water-soluble Alexa Fluor™ 568 NHS Ester (Thermo Fisher Scientific, Hudson, NH, USA) (1 μM) in TNM-FH medium with 10% FBS medium for 30 min. The medium was removed and the cells were washed with PBS three times followed by addition of fresh TNM-FH medium with exosome-depleted FBS and cultured for 6 h. The culture medium was then collected and subjected to exosome isolation using an exosome isolation reagent (Invitrogen). The Pxem_ZJU cells were adhered to an 8-chamber slide (Thermo Fisher Scientific) incubated with/without the fluorescently labeled medium for 18 h. DAPI was added during the last 5 min. Slides were analyzed by confocal microscopy (LSM 800, Zeiss, Jena, Germany).
7
Vitreous Exosome Isolation Protocol
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Approximately 200 µL of vitreous was processed for further experiments after routine diagnostics. Vitreous was spun in a centrifuge at 10,000 g for 15 minutes to remove cellular debris and immediately filtered with 0.22 µm filter to remove larger sized EVs. Exosome isolation reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate EVs from the vitreous according to manufacturer's instructions. Briefly, 100 µL of reagent was added to 100 µL vitreous and incubated at 4°C overnight (∼12 hours) to precipitate EVs and spun in a centrifuge at 10,000 g for 90 minutes to pellet down smaller EVs. The pellet was resuspended in 100 µL of 1X PBS, and remaining vitreous and isolated EVs were immediately transferred to −80° C until further processing.
8
Exosome Isolation from Cell Culture Supernatant
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The cell culture conditioned medium sample (7 mL) was transferred to a fresh tube, and 3.5 mL of exosome isolation reagent (Invitrogen/Thermo Fischer Scientific Inc., #4478359) was added and mixed thoroughly by pipetting up and down. The solution was incubated overnight at 4 °C. The following day, the samples were centrifuged at 10,000× g for 60 min. The pellet was then resuspended in 200 µL DPBS and stored at 2–8 °C for a maximum of up to seven days before downstream analysis or further purification by the immunomagnetic bead method.
9
Isolation and Purification of HUVEC Exosomes
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HUVEC was taken from the proximal part of the infant umbilical cord in cesarean section after the parents agreed and proved by informed consent. After being cut proximally, the umbilicus was cleaned from blood by venipuncture. After the venipuncture, within 24 hours the umbilicus should arrive at the laboratory. The umbilicus was cannulated and cleaned from the left blood with 0.2% buffer and collagenase, and then incubated for 15 minutes at 37°C. The endothelial and subendothelial layers were collected using a centrifuge and washed with PBS three times. The cultures were placed on discs and allowed to grow for three days. After fibroblastoid formation, the cells were subcultured by the warm trypsin method.
The HUVEC secretome was transferred into a sterile tube and Invitrogen ® exosome isolation reagent was added. Samples were incubated overnight at 2–80°C and centrifuged. After being centrifuged, the supernatant was removed, and the pellet at the bottom of the tube was left. The pellets were suspended with PBS and purified by the affinity method. The exosome was isolated at 2–80°C for a week
The HUVEC secretome was transferred into a sterile tube and Invitrogen ® exosome isolation reagent was added. Samples were incubated overnight at 2–80°C and centrifuged. After being centrifuged, the supernatant was removed, and the pellet at the bottom of the tube was left. The pellets were suspended with PBS and purified by the affinity method. The exosome was isolated at 2–80°C for a week
10
Exosome Isolation from Serum Samples
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Exosomes were isolated using the exosome Isolation Reagent (4478360, InvitrogenTM, Thermo Fisher Scientific®, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, serum samples were centrifuged at 2000 × g for 30 minutes to remove cell and debris. The serum samples then were filtered through 0.22 μm syringe filter (Millipore). 0.2 volumes of total exosome Isolation Reagent were added to serum (40 μL of isolation reagent to 200 μL of serum) and mixed the serum-reagent mixture by vortexing. The samples were incubated for 30 minutes at room temperature. After incubation, the samples were centrifuged at 10,000 × g for 10 min at 4°C. The exosome pellet was retrieved after removal of the supernatant and resuspended in 100 μL PBS for downstream analyses.48–50 (link)
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