Pe conjugated donkey anti rabbit igg antibody

For nanoFCM, a Nano Analyzer (NanoFCM Co. Ltd., Nottingham, United Kingdom) equipped with a 488 nm laser, was calibrated using 200 nm polystyrene beads (NanoFCM Co.) with a defined concentration of 2.08 × 108 particles/ml, which were also used as a reference for particle concentration. In addition, monodisperse silica beads (NanoFCM Co. Ltd.) of four different sizes served as size reference standards to calibrate the size of EVs. Freshly filtered (0.1 µm) 1 × PBS was analyzed as background signal and subtracted from the other measurements. Each dot plot was derived from data collected approximately 4,000 events with a sample pressure of 1.0 kPa. For immunofluorescence staining, the following antibodies were used (BioLegend): FITC-conjugated mouse anti-human/canine CD9 antibody (clone HI9a), and anti-human Ecad, with secondary PE-conjugated donkey anti-rabbit IgG antibody (clone Poly4064); as isotype controls, FITC-conjugated mouse IgG1, κ (clone MOCP-21), and PE-conjugated donkey anti-rabbit IgG antibody (clone Poly4064); 1 ng/µl of each antibody in 50 µL 1 × PBS. After removing antibody aggregates by centrifugation at 12,000× g for 10 min, the supernatant was added to 5 × 108 purified EVs, followed by incubation for 90 min at 25°C under constant shaking. Stained EV were diluted 1:100 in 1xPBS for NanoFCM analysis.

Primary antibodies used are listed in Table S4. Secondary antibodies were as follows: goat anti-rabbit-IgG conjugated to Alexa Fluor 488, goat anti-mouse-IgG conjugated to Alexa Fluor 488, goat anti-mouse-IgG conjugated to Texas Red, goat anti-mouse-IgG conjugated to Alexa Fluor 568, donkey anti-rabbit-IgG conjugated to Alexa Fluor 594, goat anti-rabbit-IgG conjugated to Alexa Fluor 647, and goat anti-mouse-IgG conjugated to Alexa Fluor 647 (Invitrogen), PE-conjugated donkey anti-rabbit-IgG antibody (Biolegend #406421), stabilized HRP-conjugated goat anti-mouse-IgG and HRP-conjugated goat anti-rabbit-IgG (Bio-Rad). A conjugated streptavidin-HRP (1:1000; GE Healthcare #RPN1231) antibody was used for detection of biotinylated proteins. Calyculin A (#9902) was from Cell Signaling Technology and nocodazole from Sigma-Aldrich.