Fig 1.
MP analysis by flow cytometry and electron microscopy.
Calibration of Navios cytometer using 0.1, 0.3, 0.5, and 0.9 μm beads (Biocytex). The cytometer is able to differentiate the 4 different populations of beads (A-B). We can delimit the gate in accordance with size to study MPs (0.3–0.9 μm) (C). Characteristic elongated shape of MPs repartition limited in a gate of 0.3–0.9 μm. (D). Labeling of platelet derived-MPs with CD41 and annexin-V revealed a double positive population for these markers (E). Example of 7-AAD/annexin V labeling to detect the presence of apoptotic bodies (AB). (F). Absence of contamination with AB. To monitor the presence of protein complexes (PC), lysis with 0.05% triton was used. After lysis, the positive signal disappeared (G). Scanning Electron Microscopy (SEM) shows the production of MPs by B-lymphocytes (white arrows) (H). Transmission electron microscopy (TEM) shows the structure of one MP with a characteristic lipid bilayer (white arrows) (I).
Fig 2.
MP characterization by direct flow cytometry.
A representative case for each staining is provided: for platelet-derived MPs, CD41 (A) associated with annexin-V labeling (B), similar to that found in the original cells (C). For MPs derived from other cellular sources, no antigen was detected (CD20 and CD14) (D/G) despite the expression of these markers on cells (F/I). An annexin-V positive population is present for all samples (ranging from 32% to 94%). For NK-cells, T-lymphocytes, and CLL B-cells, we observed a weak positive signal with CD56, CD3, and CD19, respectively. These signals were thus false positives, demonstrated by the use of other negative controls (Fig 3). Isotypes were used as negative controls (open line) and the tests are presented by filled histogram.
Fig 3.
Cross-labeling MPs from different cell types: non-specific labeling.
Double staining with annexin-V and specific markers was applied to four MP types (B-lymphocytes, T-lymphocytes, monocytes and NK-cells). Isotypes were used as negative controls (open line) and the tests are presented by filled histogram. Histograms of cell-derived MPs stained with CD19, CD3, CD14, and CD16 showed that non-specific labeling occurs for the majority of samples.
Table 1.
Mean fluorescence intensity ratio (MFIR) analysis on MPs by direct flow cytometry.
Fig 4.
MP characterization by latex bead technique.
We studied three types of cell-derived MPs with the latex bead technique: platelet-, BM-MSC- and B-cell-derived MPs. Beads with ‘‘washing antibody” were used as negative controls. Three different labels were applied to these MPs. (I) We confirmed the expression of CD41 on platelet-derived MPs, (G-H) and negative signal for CD90 and CD19. (C/F) Although CD41 expression was negative for B-cell and BM-MSC-derived MPs, (A-B, D-E) we observed CD90 and CD19 expression on both B-cells and BM-MSC-derived MPs, confirming false positive labeling.