Separation and isolation of CD9-positive extracellular vesicles from plasma using flow cytometry

Abstract

Extracellular vesicles (EVs) are nanosized (∼30-1000 nm) lipid-enclosed particles released by a variety of cell types. EVs are found in biological fluids and are considered a promising material for disease detection and monitoring. Given their nanosized properties, EVs are difficult to isolate and study. In complex biological samples, this difficulty is amplified by other small particles and contaminating proteins making the discovery and validation of EV-based biomarkers challenging. Developing new strategies to isolate EVs from complex biological samples is of significant interest. Here, we evaluate the utility of flow cytometry to isolate particles in the nanoscale size range. Flow cytometry calibration was performed and 100 nm nanoparticles and ∼124 nm virus were used to test sorting capabilities in the nanoscale size range. Next, using blood plasma, we assessed the capabilities of flow cytometry sorting for the isolation of CD9-positive EVs. Using flow cytometry, CD9-positive EVs could be sorted from pre-enriched EV fractions and directly from plasma without the need for any EV pre-enrichment isolation strategies. These results demonstrate that flow cytometry can be employed as a method to isolate subpopulations of EVs from biological samples.

Fig. 1. Detection and sorting of nanoparticles using the Astrios EQ flow cytometer. (A) Calibration plot using SPHERO™ Rainbow Calibration Particles with known Equivalent Number of Reference Fluorophores (ERF) value for each bead plotted against their fluorescence intensity (arbitrary units). (B) Representative image of the baseline noise detected from the Astrios EQ when running deionized water in the fluorescent FITC channel. (C and D) Megamix-Plus FSC polystyrene beads ranging from 0.1, 0.3, 0.5, and 0.9 μm triggered using 488 FSC-H and analyzed on: (C) the FITC channel and (D) 488-side scatter (488-SSC-A). (E) Gated fluorescent beads analyzed by 488-SSC-A. (F) Selection of 0.1 μm nanoparticles from the Megamix-Plus FSC beads for sorting using the Astrios EQ. (G) Sorted 0.1 μm beads analyzed by the Astrios EQ to confirm isolation of the 0.1 μm nanoparticles from the Megamix.

Fig. 2. Detection and sorting of GFP-viral particles using the Astrios EQ flow cytometer. (A) GFP virus detection using the FITC-A channel. (B) Overlay plot of the GFP virus and the background noise from the Astrios EQ. (C) 25 μL of GFP virus spiked into 100 μL of filtered plasma analyzed on the Astrios EQ. (D) Plasma, without GFP virus spike, analyzed using the fluorescent FITC-A channel. (E) Sorted GFP virus analyzed using the Astrios EQ flow cytometer. (F) GFP virus diluted in PBS and analyzed on a nanoscale flow cytometer analyzer (CytoFLEX S). (G) Sorted GFP virus analyzed by a nanoscale flow cytometer analyzer (CytoFLEX S).

Fig. 3. 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. 4. 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.