P40st swing rotor

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.

OptiPrep iodixanol density media was obtained from Abbott Diagnostics Technologies AS. The continuous 0 to 30% or 0 to 45% OptiPrep gradients were prepared in 30 mM MES–Tris (pH 6.9), 0.1 M KCl, 0.5 mM MgCl₂, 0.2 M sorbitol using a Gradient Master (BioComp Instruments, Inc.). Before loading, the vacuolar fraction was filtered (0.8 μm PC Membrane, ATTP01300, or 1.2 μm PC Membrane, RTTP02500, Merck Millipore) and the vacuolar fraction (less than 1 ml) was added to the top of 11 ml gradients and centrifuged at 72,000g for 90 min using a P40ST swing rotor (Hitachi Koki). Fractions were collected from the top using a Piston Gradient Fractionator (Model 152 BioComp Instruments, Inc). Twelve fractions (1 ml each for fraction 1–10, 0.6 ml for fraction 11, and 0.2 ml (bottom) for fraction 12) were collected. Aliquots (15 μl) of each fraction were resolved by SDS-PAGE and subjected to immunoblotting. For qRT-PCR analyses, aliquots (10 μl) of each fraction were used. For staining by Coomassie brilliant blue, the vacuolar fraction (640 μl) and AB fraction (640 μl) were precipitated with 10% trichloroacetic acid and resolved by SDS-PAGE before staining with Coomassie brilliant blue (Nacalai Tesque, 04543-64).

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.