13pet tube

For the isolation of milk sEV from BLV-infected and uninfected cattle, we followed the procedure previously described by Yamauchi et al.^{40 (link)} and Rahman et al.^{41 (link)} with slight modifications. Importantly, after defatting of milk, milk sEV were purified using acetic acid followed by sequential filtration through 1.0, 0.45, and 0.2 μm filters (GA-100, C045A047A, and C020A047A, Advantec, Tokyo, Japan). Subsequently, milk sEV were concentrated by ultracentrifugation (UC) at 100,000 × g at 4 °C for 1 h using a P42A angle rotor (Hitachi Koki, Tokyo, Japan) in a Himac CP80WX ultracentrifuge (Hitachi Koki). After the first UC, the supernatant was discarded and the pellet was resuspended with PBS up to 10 ml into a 13PET tube (Hitachi Koki) followed by another UC at 100,000 × g at 4 °C for 1 h using a P40ST swing rotor (Hitachi Koki). sEV pellet was collected and resuspended with 100 µl of PBS for further use.

Isolation and purification of milk sEVs were carried out as described previously [15 (link),16 (link)] with slight modifications. Briefly, after removing the milk fat by centrifugation at 2000× g for 20 min using a centrifuge (MX-307, Tomy Seiko, Tokyo, Japan), defatted milk was preheated at 37 °C for 10 min. For efficient isolation of milk sEVs, acetic acid was added (finally 1%), and casein was removed by centrifugation at 5000× g for 20 min. The whey was filtrated by using 1.0, 0.45, and 0.2 μm pore-size filters (GA-100, C045A047A, and C020A047A, Advantec, Tokyo, Japan). Further, milk sEVs were concentrated from the whey by ultracentrifugation (UC) at 100,000× g at 4 °C for 1 h using a P42A angle rotor (Hitachi Koki, Tokyo, Japan) in a Himac CP80WX ultracentrifuge (Hitachi Koki). The supernatant was discarded and the bottom pellet was resuspended with phosphate-buffered saline (PBS) up to 10 mL into a 13PET tube (Hitachi Koki). The UC was carried out twice again at 100,000× g at 4 °C for 1 h using a P40ST swing rotor (Hitachi Koki) and the milk sEVs pellet was resuspended with 100 µL of PBS for further use.