Figure 1
The rotor type employed during the first centrifugation step of the isolation protocol affects the MV recovery. (A) Flow chart of the differential centrifugation protocol. (B) Flow cytometry analysis (FlowJo, version 10) so of IB4/Annexin V double-stained MVs isolated from untreated (NS) and ATP-stimulated CHME-5 microglia cells adopting SW or FA rotor during the first centrifugation step. (C) Quantification of IB4/Annexin V + MVs isolated using the SW or the FA rotor in NS and ATP conditions as measured by flow cytometry. Data are presented as mean ± SEM, *p < 0.05, ***p < 0.001, ANOVA with Bonferroni post hoc test (GraphPad Prism, version 5). (D) FA to SW rotor ratio from MV quantification as shown in (C) calculated as (FA-MV number/106 cells)/(SW-MV number/106 cells). Data are reported as mean ± SEM (GraphPad Prism, version 5). (E, G) Transmission electron microscopy (TEM) and atomic force microscopy (AFM) of isolated MVs for SW and FA protocol, in NS and ATP conditions. Scale bars 200 nm. (F,H) Size distribution of isolated MVs in ATP condition determined from TEM (F, SW mean size of 155.4 ± 6.6 nm, n = 100; FA mean size of 133.8 ± 2.1 nm, n = 250) and AFM (H, SW mean size of 173 ± 11.5 nm, n = 50; FA mean size of 154.3 ± 2.8 nm, n = 300) images using Image J software.
