Vacuum manifold

EV-depleted FBS was prepared by 18 h-ultracentrifugation at 100,000g, 4 °C [31 ]. 4T1 cells were seeded at 7 × 10⁵ cells or 1.5 × 10⁶ cells per 60 mm or 100 mm cell culture dishes respectively, and cultured in the EV-depleted medium, and microvesicles were then harvested as described previously [14 (link)]. Briefly, conditioned medium was centrifuged at 600 × g for 30 min to remove cells and debris. The supernatant was centrifuged again at 2,000 × g for 30 min to remove apoptotic bodies. Microvesicles were collected by ultracentrifugation at 20,000 × g for 60 min using Optima XL-90 Ultracentrifuge and 90Ti rotor (Beckman Coulter), or refrigerated microcentrifuge 5424 R (Eppendorf) for small volumes. Supernatants were filtered through 0.22-μm membrane filters (Thermo Scientific) with pressure to remove large vesicles. Exosomes were collected by a size-based EV isolation method with modifications [32 (link)] using 50 nm membrane filters (EMD Millipore, VMWP02500) with holders (EMD Millipore, SX0002500). Briefly, holders with 50 nm membrane filters were connected to a vacuum manifold (Qiagen), and the membrane filters were first washed with 5 – 10 mL of PBS buffer by applying vacuum. Then, the remaining EVs including exosomes in the supernatant were trapped on the membranes. When ~100 μL of sample remained, the concentrated EVs were carefully collected.