Fig. 4

MSC-derived migrasomes exhibit a distinctive cell surface CD marker profile and carry intracellular vesicles. A–I Primary human MSCs were cultured on fibronectin-coated glass coverslips for 24 h before being processed for CLSM. PFA-fixed cells were permeabilized with saponin or non-permeabilized (NP) before immunolabeling for a given CD marker (A–C, E, G), specific tetraspanin proteins (TSPAN 2 and 4, F), Rab7 or α-tubulin (I) as indicated followed with the appropriate fluorophore-conjugated secondary antibody and membrane staining with fluorophore-conjugated WGA. CD166 immunolabeling shows its presence in mid- and fully formed migrasomes (B, C, arrowheads), whereas it is absent or very weakly expressed in early stage migrasomes (B). Quantification of CD166 signal in migrasomes and its comparison to cellular protein values show elevated levels of CD166 protein in migrasomes (D). The box-and-whisker plot shows data from 25 to 75th percentiles and 95% within the whiskers. Horizontal line represents the median, while each dot denotes a single migrasome. 3D render of a single migrasome presented in G highlights the presence of an intracellular pool of CD63 (H, arrowhead). J–L Transiently transfected Rab7-GFP MSCs were immunolabeled for CD63 and stained with fluorophore-conjugated WGA (white in J). A 3D render of the migrasome presented in J highlights the presence of cytoplasmic CD63 and late endosomal marker Rab7 (L, arrowhead). Cell-free, detached migrasomes are displayed (K). The images presented in H and L are excerpted from the Additional file 6: Video S5 and Additional file 7: Video S6, respectively. Scale bars, 5 μm (A–C, E–G, I–K); 2 μm (L); 1 μm (A, CD166, lower panel, H)