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.
Mla 55 fixed angle rotor
Manufactured by Beckman Coulter
Sourced in United States
The MLA-55 fixed-angle rotor is a laboratory centrifuge accessory designed for Beckman Coulter's high-speed centrifuges. It is used for the separation and isolation of biological samples, such as cells, proteins, and nucleic acids. The rotor's fixed-angle design allows for efficient sedimentation and separation of sample components based on their density and size.
Automatically generated - may contain errors
Lab products found in correlation
Optima max xp benchtop ultracentrifuge, by Beckman Coulter (1 mentions)
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)
Protease inhibitor, by Avantor (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
3 protocols using mla 55 fixed angle rotor
1
HT29 Cell Culture for Microscopy
2
Exosome Isolation from Platelet-free Plasma
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Seven mL of 0.8 μm filtered and 2× PBS-diluted platelet-free plasma samples were centrifuged at 13,200×g and 4°C for 22 min to remove microvesicles. The supernatant was filtered twice through 0.22 μm filters (pre-cleared plasma). Exosomes were pelleted with UC at 120,000×g in an MLA-55 fixed-angle rotor (Beckman Coulter, Brea, CA, US) with various conditions. Either 7 mL pre-cleared plasma was centrifuged for (a) 1h, (b) 3h, (c) 6h, (d) 14h at 4°C, or (e) for 1h at 37°C (supplemented with protease inhibitor [Amresco, Solon, OH, US]). Furthermore, in experiment (f) 7 mL 5× diluted pre-cleared plasma (ultimately 10-fold dilution, since platelet-free plasma was 2× diluted before) was centrifuged for 1h at 4°C, and in experiment (g) 1 mL pre-cleared plasma was centrifuged for 1h at 4°C. In case of experiments (a), (e), (f), and (g), supernatants after ultracentrifugation were repeatedly ultracentrifuged with the same conditions. This procedure was repeated three times. Pellets were washed once with PBS and centrifuged at 120,000×g, 4°C for 1h in case of samples that were centrifuged for 1h in the previous step or for 2h in experiments (b), (c), and (d). Samples were used fresh, since our experiments indicated that integrity of exosomes is compromised when stored for 4 weeks or longer (Fig A-C in S1 File )
3
Exosome Isolation from Human Vitreous Humor
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Differential ultracentrifugation was performed for exosome isolation from human VH samples, as described previously [23] . All centrifugation steps were performed at 4 °C. In brief, 0.5 mL of each VH sample was centrifuged at 2000×g and 10,000×g for 30 and 45 min, respectively, at 4 °C to eliminate cellular debris. The supernatant was filtered twice through 0.22 μm filters (Millipore) and then moved to new tubes. The exosomes were pelleted with ultracentrifugation at 100,000×g for 70 min in an MLA-55 fixed-angle rotor (Beckman Coulter, Brea, CA, USA). The pellet was washed in phosphate-buffered saline (PBS) to prevent protein contamination and centrifuged again at 100,000×g for 70 min. The PBS was removed, and the exosomes were resuspended in 100 µL PBS and stored at -80 °C for further analysis. A BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) was performed for the total protein of the exosomes according to the manufacturer's protocol. Quantification of exosomes was performed using electron microscopy, nanoparticle tracking analysis (NTA) and flow cytometry. Exosome counts were used to normalise the mass spectrometer (MS) intensities for each detected m/z.
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