To isolate cell-derived exosomes, myeloma cells from patients with MM and MM cell line RPMI-8226 were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 µg/mL penicillin (Gibco), and 100 U/mL streptomycin (Gibco) in a humidified atmosphere (37.5°C and 5% CO2) for 24 hours. To remove exosomes of fetal bovine serum used in culture media, it was subjected to ultracentrifugation at 100,000 g for 3 hours at 4°C. The culture medium was collected and centrifuged at 800 g for 5 min, followed by 2000 g for 10 min to get rid of lifted cells. The supernatant was filtered on a 0.1 mm pore polyethersulfone membrane (Corning) to remove cell debris and large vesicles, and then concentrated through a 100,000 Mw cut-off membrane (CentriPlus-70, Millipore, Bedford, Massachusetts, USA). The volume of supernatant was reduced from approximately 250–500 mL to less than 5 mL. The supernatant was then ultracentrifuged at 100,000 g for 1 hour at 4°C using 70Ti rotor (Beckman Coulter). The resulting pellets were resuspended in 6 mL phosphate-buffered saline (PBS) and ultracentrifuged at 100,000 g for 1 hour at 4°C using 100Ti rotor (Beckman Coulter).19 (link)
Centriplus 70
Manufactured by Merck Group
Sourced in United States
CentriPlus-70 is a centrifuge product manufactured by Merck Group. It is designed to separate and isolate different components within a liquid sample through the application of centrifugal force. The core function of the CentriPlus-70 is to facilitate the efficient separation and isolation of materials with varying densities.
Automatically generated - may contain errors
Lab products found in correlation
70ti rotor, by Beckman Coulter (6 mentions)
100ti rotor, by Beckman Coulter (5 mentions)
0.1 mm pore polyethersulfone membrane filter, by Corning (3 mentions)
0.1 mm pore polyetherrsulfone membrane filter, by Corning (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using centriplus 70
1
Isolating Myeloma Cell-Derived Exosomes
2
Exosome Isolation from A549 Cell Culture
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Exosome extraction was performed as previously described [41 (link)]. Briefly, A549 cells were cultured in serum-free DF12 medium for 24 h. Then, the culture medium was collected and centrifuged at 800g for 5 min and additional 2000g for 10 min to remove lifted cells. The supernatant was subjected to filtration on a 0.1-mm-pore polyethersulfone membrane filter (Corning) to remove cell debris and large vesicles, followed by concentration by a 100,000 Mw cutoff membrane (CentriPlus-70, Millipore). The volume of supernatant was reduced from approximately 250–500 mL to less than 5 mL. The supernatant was then ultracentrifuged at 100,000g for 1 h at 4 °C using 70Ti rotor (Beckman Coulter). The resulting pellets were resuspended in 6 mL PBS and ultracentrifuged at 100,000 g for 1 h at 4 °C using 100Ti rotor (Beckman Coulter).
3
Exosome Isolation from HepG2 Cells
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Exosome extraction was performed as previously described [22 (link)]. Briefly, HepG2 cells were cultured in serum-free DF12 medium for 24 h. Then, the culture medium was collected and centrifuged at 800g for 5 min and additional 2000g for 10 min to remove lifted cells. The supernatant was subjected to filtration on a 0.1-mm-pore polyethersulfone membrane filter (Corning) to remove cell debris and large vesicles, followed by concentration by a 100,000-Mw cutoff membrane (CentriPlus-70, Millipore). The volume of supernatant was reduced from approximately 250–500 mL to less than 5 mL. The supernatant was then ultracentrifuged at 100,000g for 1 h at 4 °C using 70Ti Rotor (Beckman Coulter). The resulting pellets were resuspended in 6 mL PBS and ultracentrifuged at 100,000g for 1 h at 4 °C using 100Ti Rotor (Beckman Coulter). In the experiments involving HepG2 exosomes, we use PBS as a negative control.
4
Isolation and Characterization of MSC-derived Small Extracellular Vesicles
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We followed the MISEV 2018 guidelines to isolate and identify MSC-sEV. Briefly, after MSCs reached 80–90% confluence, the serum-free medium was added for 48 h to avoid contamination of vesicles from serum. The conditioned medium was collected and centrifuged 800 g for 30 min and additional 3000 g for 30 min to remove cells and debris. The supernatant was then subjected to a 0.1-mm-pore polyetherrsulfone membrane filter (Corning) filtration to eliminate cell debris and large vesicles, followed by a 100,000-Mw cutoff membrane concentration (CentriPlus-70, Millipore). The supernatant volume was reduced to less than 5 mL from approximately 250–500 mL. Using the 70Ti Rotor, the supernatant was then ultracentrifuged at 110,000 g for 2 h at 4 °C (Beckman Coulter). The resulting pellets were resuspended with PBS and ultracentrifuged with 100 Ti Rotor for 1 h at 110,000 g at 4 °C (Beckman Coulter). We used PBS buffer as a negative control in the experiments involving hBMSC-sEV.
5
Isolation and Identification of MSC-derived Extracellular Vesicles
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We followed the MISEV 2018 guidelines to isolate and identify MSC-sEV53 (link),54 (link). Briefly, after MSCs reached 80–90% confluence, the serum-free medium was added for 48 h to avoid contamination of vesicles from serum. The conditioned medium was collected and centrifuged 800 g for 5 min and additional 3000 g for 10 min to remove cells and debris. The supernatant was then subjected to a 0.1-mm-pore polyetherrsulfone membrane filter (Corning) filtration to eliminate cell debris and large vesicles, followed by a 100,000-Mw cutoff membrane concentration (CentriPlus-70, Millipore). The supernatant volume was reduced to less than 5 mL from approximately 250–500 mL. Using the 70Ti Rotor, the supernatant was then ultra-centrifuged at 110,000 g for 2 h at 4 °C (Beckman Coulter). The resulting pellets were resuspended with PBS and ultra-centrifuged with 100 Ti Rotor for 1 h at 110,000 g at 4 °C (Beckman Coulter). We used phosphate-buffered saline (PBS) as a negative control in the experiments involving MSC-sEV.
6
Exosome Isolation from Mesenchymal Stem Cells
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Exosome extraction was performed as previously described. Briefly [22 (link)], MSCs were cultured in serum-free DMEM/F12 medium for 48 h. The culture medium was then collected and centrifuged at 800 g for 5 min and an additional 2000 g for 10 min to remove lifted cells. The supernatant was subjected to filtration on a 0.1 mm pore polyethersulfone membrane filter (Corning) to remove cell debris and large vesicles, followed by concentration with a 100,000-Mw cutoff membrane (CentriPlus-70; Millipore). The volume of the supernatant was reduced from approximately 250-500 mL to less than 5 mL. The supernatant was then ultracentrifuged at 100,000 g for 1 h at 4 °C using the 70Ti rotor (Beckman Coulter). The resulting pellets were resuspended in 6 mL PBS and ultracentrifuged at 100,000 g for 1 h at 4 °C using the 100Ti rotor (Beckman Coulter).
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