Two million MSC were seeded in cell culture flasks with 15 ml of complete medium depleted from fetal bovine serum (FBS)-derived EVs (11 (link)). To deplete medium from FBS-EVs, 20% FBS complete medium (αMEM +1% P/S +2 mM L-Glutamine) was ultracentrifuged at 100,000 × g for 16 h in polypropylene ultracentrifugation tubes (Beckman coulter, Brea, CA). The supernatant was collected and filtered through a 0.22 μm filter (Sarstedt, Germany) to sterilize the medium, which was finally diluted with αMEM medium to the final concentration of 10% FBS for cell culture. After 48 h, the medium was collected and centrifuged at 400 × g and 2,000 × g to eliminate cells and cell debris, respectively, to obtain MSC-conditioned medium (CM).
Polypropylene ultracentrifugation tubes
Manufactured by Beckman Coulter
Polypropylene ultracentrifugation tubes are designed for high-speed centrifugation applications. They are made of polypropylene, a durable and chemically resistant material. These tubes are intended to withstand the high centrifugal forces generated during ultracentrifugation procedures.
Automatically generated - may contain errors
Lab products found in correlation
0.22 μm filter, by Sarstedt (1 mentions)
Human platelet lysate, by Lonza (1 mentions)
Heat inactivated fbs, by Lonza (1 mentions)
0.22 m filter, by Sarstedt (1 mentions)
Plasmocin, by InvivoGen (1 mentions)
Texmacs, by Miltenyi Biotec (1 mentions)
α mem, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
2 protocols using polypropylene ultracentrifugation tubes
1
Generating FBS-Depleted MSC-Conditioned Medium
2
Optimized Culture Medium for MSC and T Cells
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Complete culture medium was composed of α-MEM (Sigma Aldrich) or TexMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) medium supplemented with 2mM L-Glutamine (Sigma Aldrich), 100U/ml Penicillin (Cepa S.L., Madrid, Spain), 100µg/ml Streptomycin (Normon Laboratories S.A., Madrid, Spain) and 10% (v/v) Heat Inactivated-FBS or Human platelet lysate (Lonza, Basel, Switzerland) for MSC and T cell culture, respectively. Plasmocin (5 µg/mL; Invivogen) was added for MSC culture.
Culture medium was depleted of bovine/human EVs by ultracentrifugation of 2x complete medium in polypropylene ultracentrifugation tubes (Beckman coulter, Brea, CA) at 100,000g for 16 h (SW28 rotor, 28000 rpm, adjusted k-Factor= 253.96). The supernatant was collected and filtered through a 0.22 µm filter (Sarstedt, Germany) to sterilize the medium, which was finally diluted to 1x working concentration with αMEM/TexMACS medium alone for cell culture.
Culture medium was depleted of bovine/human EVs by ultracentrifugation of 2x complete medium in polypropylene ultracentrifugation tubes (Beckman coulter, Brea, CA) at 100,000g for 16 h (SW28 rotor, 28000 rpm, adjusted k-Factor= 253.96). The supernatant was collected and filtered through a 0.22 µm filter (Sarstedt, Germany) to sterilize the medium, which was finally diluted to 1x working concentration with αMEM/TexMACS medium alone for cell culture.
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