Magcapture exosome isolationkit ps version 2

EVs were
separated from pooled human serum using the MagCapture Exosome Isolation
Kit PS version 2 (# 290-84103 Fujifilm WAKO Pure Chemical Corporation)
with the KingFisher Flex System (Thermo Fisher Scientific). Briefly,
200 μL of serum was added to 300 μL of Tris-buffered saline
and centrifuged at 1200 × g for 20 min at 4
°C. The supernatant was filtered through a 0.45-μm filter
(# 7820-11001 FastRemover for Protein) using Positive pressure Resolvex
M10 96 (TECAN). EVs were isolated from filtered serum using the KingFisher
Flex System. The EVs were eluted with 100 μL of elution buffer
for proteomics analysis. The program of KingFisher Flex for the EV
isolation process is shown in Table S3.

EVs were separated from human plasma and serum using the MagCapture Exosome Isolation Kit PS version 2 (# 290-84103 Fujifilm WAKO Pure Chemical Corporation). Briefly, 150 μl of plasma and serum were added to 350 μl of Tris-buffered saline (TBS) and centrifuged at 1,200 g for 20 min at 4°C. The supernatant was filtered through a 0.45-μm Spin-X centrifuge tube (# CLS8162 Corning), and then 5.0 μl of 1 U/μl heparin sodium solution (# 085-00134 Fujifilm WAKO Pure Chemical Corporation) was added only to the EDTA plasma samples to maintain the anticoagulant effect instead of EDTA. The EVs were eluted with 100 μl of elution buffer, and 80 μl of eluate was used for proteomics analysis.