fig4

Figure 4. EV phenotype in pooled secretomes. (A) Nanoparticle tracking analysis of pooled secretomes obtained after cultivating BMSCs in F, P, and S/X media; (B) Flow cytometer analysis of FITC-fluorescent nanobeads (100 - 160 - 200 - 240 - 300 - 500 - 900 nm) to ensure correct visualization of particles within the study range (< 100 nm up to 1 µm); (C) Flow cytometer detection of CFSE-labeled EVs compared to unstained EVs. Only unstained EVs in F secretomes are shown for readability. A representative plot is shown; (D and E) EVs in the different media were positive for EV markers CD63 and CD81, and MSC lineage markers CD73 and CD90. CD9, another EV marker, was almost absent, while CD44 (an MSC-lineage marker) was dim. Only unstained CFSE-EVs from the F condition are visualized for readability. A representative plot per Ab is shown; (F) Violin plot of CD44 positivity, N = 3 pools (each from 8 donors). Median and quartiles are shown as dashed and dotted lines, respectively. BMSCs: Bone marrow-derived mesenchymal stromal cells; F: fetal bovine serum; P: platelet lysate; S/X: serum/xeno free; EVs: extracellular vesicles; CFSE: carboxyfluorescein succinimidyl ester; MSC: mesenchymal stromal cells. ^***P-value ≤ 0.001.