HT29 human colorectal cancer cells (ATTC_HTB-38) were grown in triplicates for 24 h on 8-well Nunc Lab-Tek (177,402, Nagle Nunc, Rochester, USA) for confocal microscopy and 8-well Ibitreat microscopy chamber (Ibidi GmbH, Martinsried, German) for STED microscopy (initial cell number: 5000 cell/300 µl/well). For electron microscopy (EM) and DAB immune EM, we cultured the cells on Lab-Tek chamber (initial cell number: 8000 cell/300 µl/well) for 72 h in RPMI 1640 medium (Biosera, Ringmer, UK) supplemented with 2 mM L-glutamine (Merck-Sigma-Aldrich, Darmstadt, Germany), 80 mg/2 ml gentamycin (Sandoz) and 10% sEV-depleted FBS. To preserve the MVB-like sEVCs, we decanted the culture medium carefully from the cultures, instead of aspirating it with a pipette. The sEV depletion was performed by ultracentrifugation of foetal bovine serum (Merck-Sigma-Aldrich, Darmstadt, Germany) in an Optima MAX-XP Benchtop Ultracentrifuge with an MLA-55 fixed-angle rotor (Beckman Coulter, Brea, USA) at 120,000 g for 16 h.
Optima max xp benchtop ultracentrifuge
Manufactured by Beckman Coulter
Sourced in Germany, United States
The Optima MAX-XP Benchtop Ultracentrifuge is a compact, high-performance centrifuge designed for laboratory use. It is capable of reaching speeds up to 120,000 rpm and can generate centrifugal forces up to 803,000 x g. The Optima MAX-XP is equipped with a variable-speed drive and can accommodate a wide range of rotor types to enable diverse applications.
Automatically generated - may contain errors
Lab products found in correlation
Ibitreat microscopy chamber, by Ibidi (1 mentions)
L glutamine, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Gentamycin, by Sandoz (1 mentions)
Rpmi 1640 medium, by Biosera (1 mentions)
Mla 55 fixed angle rotor, by Beckman Coulter (1 mentions)
Lysosome isolation kit, by Merck Group (1 mentions)
3 protocols using optima max xp benchtop ultracentrifuge
1
HT29 Cell Culture for Microscopy
2
Isolation of Mouse Cerebella Lysosomes
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Fresh cerebella were dissected from mice that were starved overnight and killed the next morning. Then lysosome isolation by subcellular fractionation from the mice cerebella was performed with a lysosome isolation kit (Sigma-Aldrich) according to the manufacturer's manual. After a discontinuous iodixanol gradient centrifugation using Optima MAX-XP Benchtop Ultracentrifuge (Beckman Coulter) with MLS-50 rotor at 150,000 × g and 4°C for 4 h, the sample was divided into ten fractions (0.5 ml each) for further biochemical analyses.
3
Isolation of Extracellular Vesicles from Nucleus Pulposus
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Nucleus pulposus grafts were incubated in the serum-free culture media (Ham’s F-12 nutrient mixture) at 37 °C in a humidified 5% CO2 incubator for 3 h. After incubation, media were centrifuged at 300g for 10 min and then at 1000g for 10 min to remove cells and other larger contaminants. Media were then centrifuged for 45-min at 10,000g. Pellets were discarded and supernatants were passed through a 0.45-µm filter. Supernatants were ultracentrifuged (Optima MAX-XP Benchtop Ultracentrifuge, Beckman Coulter, Brea, CA, USA) at 100,000g for 90 min to pellet ELVs. The pellet was washed once with PBS and centrifuged again at 100,000g for another 90 min. The ELV pellets were resuspended in PBS and stored at −21 °C for further analyses. As previously described [32 (link)], all centrifugation steps were performed at 4 °C.
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