Amicon ultra 4 10 kda nominal

The culture supernatants of EO771 cells at approximately 60–70% confluence were harvested after 16 h conditioning in serum-free media (11 (link)). Exosomes were isolated as previously described (6 (link)). Briefly, cells and debris were cleared from the supernatant by centrifugation (500 g, 10 min), followed by filtration using 0.22 μm filters (Merck Millipore). Cell-free supernatants were concentrated by ultrafiltration through Centricon Plus-70 Centrifugal Filter (100 kDa; Merck Millipore), spun at 3,500 g at 4°C. Exosomes were subsequently purified by overlaying concentrated samples on qEV size exclusion chromatography columns (Izon Science Ltd.) followed by elution with PBS. Finally, the elute from qEV columns were concentrated using Amicon Ultra-4 10-kDa nominal molecular weight centrifugal filter units (Merck Millipore) to a final volume of approximately 200 µL.

Cells were seeded into 150 × 25 mm dishes at a concentration of 1 × 10⁶ cells/dish for EO771 and PyMT cell lines, and 1.5 × 10⁶ cells/dish for MDA-MB-231 and MCF-7 cell lines. After overnight adhesion, cells were washed with Dulbecco’s phosphate buffered saline (PBS; Gibco™, cat. #14190-144) and conditioned in EV-depleted 5% FBS-containing DMEM (15 ml/dish). Conditioned media were collected from 48-h cell cultures (cell viability > 95%), followed by centrifugation (500 x g; 10 min) and filtration (0.22 µm) to remove dead cells and large debris. Exosomes were pelleted by ultracentrifugation at 100,000 x g for 90 min at 4 °C and washed once in PBS. Further purification of exosomes was performed by overlaying exosome suspensions on qEV size exclusion chromatography columns (Izon Science Ltd) followed by sample concentration in Amicon Ultra-4 10-kDa nominal molecular weight centrifugal filter units (Merck Millipore) to a final volume of 200 µl for further analysis. For human plasma samples, 1 ml of processed plasma was directly overlaid onto qEV size exclusion columns (Izon Science Ltd) followed by sample concentration to a final volume of 100 µl.