EVs were lysed in Laemmli sample buffer (252 mM Tris-HCl, 40% glycerine, 8% sodium dodecyl sulphate, 0.04% bromephenol blue, 4% β-mercaptoethanol, pH 6.8), boiled, run in 10% (w/v) polyacrylamide gels and transferred to nitrocellulose membranes. After blocking for 1 h in PBS containing 5% ovalbumin and 0.1% (w/v) Tween 20, blots were incubated with anti-lactoferrin polyclonal antibody [39 (link)] in 1:1000 dilution or anti-β-actin mAb (clone AC-74, both from Merck, Darmstadt, Germany) in 1:10,000 dilution or with anti-MPO monoclonal antibody (clone 2C7, Cell Signalling Technologies, Leiden, The Netherlands) in 1:500 dilution in 5% ovalbumin. Bound antibody was detected with enhanced chemiluminescence using horseradish peroxidase linked whole anti-rabbit-IgG (from donkey) or anti-mouse-IgG (from sheep) secondary antibodies (both from GE Healthcare, Little Chalfont, Buckinghamshire, UK) used in 1:5000 dilution.