The conditioned medium described above was collected and centrifuged at 2000 × g for 10 min and then at 10,000 × g for 30 min. The resulting supernatant was filtered through a 0.2-µm membrane to remove cells and cellular debris, as only EVs < 0.2 µm, representing exosomes and smaller MVs, were the focus of this study, and then stored as a culture supernatant at − 80 °C. To prepare EVs, the MCF7 culture supernatant was thawed and centrifuged at 100,000 × g for 70 min at 4 °C using an Optima TLX equipped with a TLA100.3 fixed-angle rotor (Beckman Coulter, Miami, FL, USA). The supernatant was discarded, and the pellet was resuspended in phosphate-buffered saline (PBS, pH 7.4). The mixture was re-centrifuged at 100,000 × g for 70 min at 4 °C and the pellet was resuspended in PBS.
Tla100.3 fixed angle rotor
Manufactured by Beckman Coulter
Sourced in United States
The TLA100.3 fixed-angle rotor is a laboratory equipment designed for high-speed centrifugation. It is compatible with Beckman Coulter ultracentrifuges and is used to separate and isolate biological samples based on their density and molecular weight.
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4 protocols using tla100.3 fixed angle rotor
1
Isolation and Purification of Extracellular Vesicles
2
Polysome Profiling of HCT116 Cells
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Metabolically recovering (ND to NR) HCT116 cells (4 × 108) were washed in PBS containing 100 μg/mL cycloheximide twice. Total cell extracts were suspended in 2.5 mL polysome buffer (20 mM Tris, 10 Mm MgCl2, 300 mM KCl, 10 mM dithiothreitol, 100 units/mL RNasin, 100 μg/mL cycloheximide, protease and phosphatase inhibitors, pH 7.4) and gently sheared 4× using a 26-gauge needle. Total cell extracts were centrifuged (20,000 × g 30 min) to pellet cell debris. The resulting cell extracts were layered over a 20% sucrose cushion (polysome buffer + 20% w/v sucrose) and ultracentrifuged (Beckman Coulter TLA100.3 Fixed-Angle Rotor, 149,000 × g 2 h). The supernatant containing the non-ribosomal fraction was later assessed using western blotting. To isolate polysome-bound mRNAs, cell extracts were layered over a 40% sucrose cushion (polysome buffer + 40% w/v sucrose) and the ribosomal pellet was collected for RNA extraction and qRT-PCR35 (link).
3
Isolation and Purification of Exosomes
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2 mL aliquots were processed according to a protocol adapted from Théry et al.10 (link). To remove large debris, BT474 media was centrifuged at 2,000 × g for 20 minutes and the pellet discarded. The supernatant was centrifuged at 10,000 × g for a further 30 minutes and the resulting pellet discarded. Samples were transferred to 3.5 mL polycarbonate ultracentrifuge tubes (Beckman Coulter, USA) and centrifuged for 70 minutes at 4°C and 100 000 × g in a TLA100.3 fixed angle rotor (Beckman Coulter, USA). The resulting pellets were resuspended in 1 mL PBS and transferred to 1 mL polycarbonate ultracentrifuge tubes (Beckman Coulter, USA). To concentrate the exosomes, samples were centrifuged at 4°C and 100 000 × g for 60 minutes in a TLA120.2 fixed angle rotor (Beckman Coulter, USA). The resulting pellet was resuspended in 100 µL PBS and stored at 4°C. The purification process for liposomes was identical with omission of the initial centrifugation steps for removal of debris.
4
Cell Surface Protein Isolation and Biotinylation
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Media was removed from the fibroblasts before following the protocol from the Cell Surface Protein Isolation Kit (#89881). In summary, cells were briefly washed twice with cool PBS (12.5 mM sodium phosphate dibasic dodecahydrate, 154 mM NaCl, pH 7.4) before adding 5 ml of the prepared labeling solution, sulfo-NHS-SS-Biotin EZ-link (0.5 mg/ml in PBS) for 30 min on ice with gentle agitation to ensure even coverage (Figure 1 ). Quenching buffer solution was added to quench the reaction for 5 min. Cells were then briefly washed four times with 5 ml of PBS before adding extraction buffer, either 1 ml of 1.5% (w/v) SMA, 1× protease inhibitor cocktail in PBS, or 1 ml of RIPA buffer (1% Triton X-100, 0.2% SDS, 1% sodium deoxycholate in PBS), and 1× protease inhibitor cocktail for 5 min at 4°C. In some experiments, 1 ml of 3% (w/v) SMA, 1× protease inhibitor cocktail in PBS was used as the extraction buffer. Cell culture dishes were scraped with a cell scraper, and extracts were transferred to a microfuge tube. Extracts were rotated for a further 30 min at room temperature (20°C) and then centrifuged using a Beckmann Coulter TLA-100.3 fixed-angle rotor at 100,000 rpm for 30 min at 4°C. Supernatants were collected, and pellets were resuspended in 1 ml of PBS for further analysis.
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