Figure 3
EV markers detection by flow cytometry and Western blot analysis.
(A) MFI values of the tetraspanins CD9, CD63 and CD81 in the SEC-pooled fractions, PEG precipitate and PROSPR product by bead-based flow cytometry. Bars represent mean+/−SD of four independent experiments. Differences with *P < 0.05 and **P < 0.005, were considered statistically significant by One-way ANOVA, Tukey test. (B) Western blot analyses of CD5L and LGALS3BP proteins from SEC isolated EVs, PEG and PROSPR products. SEC-EVs were concentrated using a 100 kDa-Amicon Ultra and dehydrated in a vacuum concentrator. Three different PROSPR concentrating preparations were loaded. First, the supernatant from PROSPR protein precipitation was dehydrated in a vacuum concentrator (miVac Quattro) at room temperature and resuspended in 1 mL as originally reported (i). To further concentrate the sample, (i) was then ultrafiltered as in SEC samples and dehydrated to 30 μL of final volume (ii). Alternatively, the 5000× g supernatant was directly concentrated using the 100 kDa-Amicon Ultra (Millipore), reaching a final volume of 100 μL (iii). (C) Ponceau S Staining of the blotting membrane.
