EV loading on aldehyde/sulphate latex beads

EVs (30 µg) were incubated with 1 µl aldehyde/sulphate latex beads (Invitrogen) (total volume, 500 µl PBS) for 15 min (room temperature) with continuous rotation. The reaction was stopped using 100 mM glycine and 2% BSA in PBS for 30 min (room temperature) with continuous rotation. Beads were centrifuged at 5000 × g (2 min), washed again with 300 µl PBS, permeabilized (0.2% TX-100 in PBS for 5 min), blocked (0.2% TX-100, 10% BSA in PBS for 10 min), washed and incubated with anti-MKLP1 antibody (1:50 dilution in 0.2% TX-100/2% BSA in PBS for 1 h) at room temperature with continuous rotation. Beads were washed 3× and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) at 1:100 dilution in 0.2% TX-100/2% BSA in PBS for 20 min under continuous rotation. Beads were washed 3×, resuspended in PBS and imaged using Zeiss AxioObserver Z1 microscope (Zeiss).