TIM-4 affinity purification of EVs

Prior to EV isolation, cells were sub-cultured to 20, 10 cm dishes for a total culture volume of 200 ml. Cells were incubated in serum-free media for 24 h before isolation. TIM-4, a phospholipid phosphatidylserine present on the surface of the extracellular vesicle, can be used to isolate EVs from the serum-free media. To isolate EVs by the TIM-4 affinity method, a MagCapture Exosome Isolation Kit PS (Wako, Japan) was used according to the manufacturer’s instructions. In brief, 0.6 mg of streptavidin magnetic beads, bound with 1 μg of biotinylated mouse Tim4-Fc, was added to 10K filtered supernatant and the mixture was rotated overnight at 4 °C. The beads were washed three times with 1 ml of washing buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.0005% Tween20, 2 mM CaCl₂), and the bound EVs were eluted with elution buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA).