Fig 1: Identification and isolation of CD9-positive EVs from plasma using flow cytometry. (A) Representative image of CD9-positive population from isolated EVs on the Astrios EQ and its respective isotype control. n = 2. (B) Representative image of CD9-positive population from filtered plasma on the Astrios EQ and its respective isotype control. n = 2. (C) Representative image of CD9-positive population from isolated EVs on the CytoFLEX S, its isotype control, and lysed with 1% Triton-X 100. n = 2. (D) Representative image of CD9-positive population from filtered plasma on the CytoFLEX S, its isotype control, and lysed with 1% Triton-X 100. n = 2. (E) CD9 immunostained plasma gated into three different regions followed by sorting of each region. (F) Sorted regions of interest (R1, R2, R3) analysed and characterized on the CytoFLEX S by CD9 and CD41 immunostaining.
Fig 2: Analysis and characterization of CD9 sorted populations. Flow cytometer analyser (CytoFLEX S) analysis of sorted EVs from: (A) SEC isolated EV fractions from plasma and (B) 0.8 μm filtered plasma. (A and B) CD9-positive and negative sorts analysed by CD9 immunostaining. EV characterisation performed by CD9 and CD41 dual immunostaining. EV lysis by 1% Triton-X 100. n = 2. (C) Quantification of CD9-positive EV events, by nanoscale flow cytometry, from flow cytometry sorts using before and after concentration. n = 2. (D) Representative flow plots of CD9-positive EV populations post-concentration. (E) Quantification of CD9-positive EV events from flow cytometry plasma sort before and after concentration with 10 μg mL−1 BSA. n = 3. (F) Representative plots of sorted CD9-positive EVs from plasma before concentration and post-concentration with 10 μg mL−1 BSA. n = 2. (G and H) Dot blot for CD9 (n = 2) and CD41 using (G) SEC isolated EVs and (H) flow cytometry isolated CD9-positive EVs post-concentration with 10 μg mL−1 BSA. (I and J) Scanning transmission electron microscopy images of EVs isolated by (I) SEC and (J) EVs isolated by flow cytometry sort of CD9-positive events, post-concentration with 10 μg mL−1 BSA. (K and L) Nanoparticle tracking analysis on sorted CD9-positive EVs from plasma post-concentration plus 10 μg mL−1 BSA from two different plasma samples.
Supplier Page from Abcam for Anti-CD41 antibody [MEM-06], prediluted (PE)