a, Exosome-isolation protocol from in vitro or in vivo sources. Exosomes are isolated using a multistep centrifugation procedure including a 0.22-μm filtration step. b, Western blot of actin, ARF6 and CD9 following each sequential centrifugation step during exosome isolation. c, Electron-microscopy (EM) quantification of vesicles 80–150 nm and greater than 150 nm in size; n = 80 images. d, EM negative staining of exosome fractions. Arrows indicate exosomes and protein aggregates. e, Representative EM images of the exosome fraction, and zoomed insets with arrows indicating the single membranes of exosomes. f, Gating strategy and representative flow-cytometry plots from nt shRNA and ATG16L1 KD samples of six independently repeated experiments. Exosomes were stained with antibodies against CD9, CD63, CD81 and ADAM10. Exosomes were concurrently labelled with PKH67, a lipid-membrane-incorporating dye. FSC, forward scatter; SSC, side scatter. Measurements were taken from distinct samples and graphs show means ± s.e.m. c, Two-tailed, unpaired t-test with Welch’s correction compared with PBS controls.