Background
Extracellular vesicles (EVs) are a highly heterogenous group of cell-derived membrane structures 1. Based on their biogenesis, EVs can be classified into different subtypes including exosomes and microvesicles 2. To date, there is no specific surface marker to distinguish between the different EV subtypes. Furthermore, given cells may release differently assembled EV subtypes 3–6.
EVs contain a variety of bioactive molecules such as proteins, nucleic acids, and lipids. Their composition presumably depends on the cell of origin and its physiological condition 7. Therefore, EVs have attracted broad interest as intercellular communication vehicle, disease biomarker, and therapeutic agent 8–11. However, no standard EV protein analysis assay exists that is fast, sensitive, and yet feasible to be implemented in most laboratories for high-throughput analysis 12. Traditional methods like Western blot, typically require large sample volumes and significant processing. Flow cytometry is a powerful tool but its straightforward applicability for EV studies is hampered by the small size, low refractive index, and polydispersity of exosome-sized EVs 13–15. Thus, more advanced approaches like high resolution and imaging flow cytometry have emerged 16,17. Although these methods are extremely sensitive and allow the analysis of EVs at a single level, they tend to be laborious and require specialized setups as well as extensive operator expertise 18.
Here, we report a method to rapidly analyze EV proteins from human and mouse samples in a semi-quantitative way by conventional flow cytometry 19.
Methods and Materials
Cells and cell culture
Cell lines were grown at 37 °C, 5 % CO2 in a humidified atmosphere. HEK293T cells were cultured in DMEM 10 % FBS, 1× Antibiotic-Antimycotic (Anti-Anti). Immortalized, human bone marrow-derived mesenchymal stromal cells [hTert + mesenchymal stromal cell line (MSCs)] were grown in RPMI-1640 10 % FBS, 10−6 mol/L hydrocortisone and 1× Anti-Anti. IGROV1 cells were cultured in RPMI-1640 10 % FBS and 1× Anti-Anti. PANC-1 cells were cultivated in DMEM/F12 10 % FBS. To harvest conditioned media (CM), cells were grown in OptiMEM for 48 h.
Cerebral spinal fluid (CSF) samples
CSF samples were collected from patients at Karolinska University Hospital by lumbar puncture after informed consent and approval by the local ethics committee. Samples were pre-cleared by centrifugation (400 × g for 10 min, then 2,000 × g for 10 min) and filtered through 0.22 µm syringe filters with cellulose acetate membrane.
Human blood samples
Blood samples were drawn from donors after informed consent and approval by the local ethics committee. 6 mL samples of blood were isolated in heparin and serum-separating tubes. The blood was spun at 2,500 × g for 20 min twice with the platelet poor plasma being isolated. Samples were frozen at -80 °C or kept at 4 °C and run through size exclusion chromatography columns following manufacturer’s instructions.
Mouse experiments
All experiments were approved by the local board for animal welfare. For immunomagnetic enrichment of CD81 mouse EVs, citrate plasma (100 or 300 µL) was incubated with 50 µL CD81 Exosome Isolation MicroBeads for 1 h. Magnetically labeled EVs were applied to a µ Column placed in the magnetic field of a µMACS™ Separator and washed. Labeled EVs were eluted with 100 µL PBS after removal of the column from the magnetic field and further analyzed.
Female NMRI mice were intravenously injected with 2 × 1011 hTert + MSC-EVs in 100 µL PBS. Blood was sampled by heart puncture 1 min after injection and collected into PST-tubes following manufacturer’s instructions. Samples were depleted from cells by centrifugation at 2,000 × g for 10 min. After filtration, 120 µL plasma was transferred to the MACSPlex Exosome assay.
EV isolation and enumeration
For the analysis of cell culture-derived EVs, EVs were prepared from supernatants of HEK293T cells, MSCs, IGROV1, or PANC-1 respectively. Cells and larger debris were depleted by centrifugation (10 min at 500–900 × g, then 10–20 min at 2,000 × g). Subsequently, samples were filtered through 0.22 μm filters and EVs were concentrated by ultracentrifugation (2 × 110,000 × g). Sample input of EVs was determined by nanoparticle tracking analysis (NTA).
EV analysis with the multiplex bead-based platform
120 µL samples were loaded onto 96-well 0.22 µm filter plate. To each well 15 µL of MACSPlex Exosome Capture Beads were added and plates were incubated on orbital shaker overnight (14–16 h) at 450 rpm at room temperature. Subsequently, the beads were washed in PBS and resuspended in 135 µL of MPB. EVs bound by capture beads were stained for 1 h at room temperature with 15 µL of MACSPlex Exosome Detection Reagent cocktail compromising CD9/CD63/CD81-APC antibody conjugates. After staining, beads were washed and analyzed by flow cytometry.
Flow cytometry analysis
Flow cytometric analysis was performed with a MACSQuant Analyzer 10 flow cytometer. Samples were mixed before 70–100 µL were loaded to and acquired by the instrument, resulting in approximately 7,000–12,000 single bead events per well. Data were exported to comma separated files, which were subsequently imported into MATLAB for further analysis and visualization.
Results
A multiplex bead-based assay to analyze EVs using flow cytometry
The multiplex assay comprises 39 bead populations distinguishable by flow cytometry. Each of these capture bead populations is coupled to an antibody specifically targeting one of 37 exosomal surface epitopes or two isotype negative controls. After incubation with EV-containing sample, the EVs are stained with a detection reagent, e. g., a cocktail of APC-conjugated antibodies against the tetraspanins CD9, CD63, and CD81, which are commonly expressed on EVs. Consequently, sandwich complexes are formed between the i) capture bead, ii) EVs, and the iii) detection reagent. Then, a semi-quantitative analysis of EV surface markers is performed by flow cytometry resulting in a characteristic surface profile of an EV sample (fig. 1).
