Everything about Exosomes Isolation using magnetic capture Beads

Exosomes are extracellular vesicles (EVs) in the approximate size range of 40 to 150nm in diameter and they are present in almost all body fluids. They contain some very common markers sus as CD9, CD63, and CD81, which makes them very interesting in research and diagnosis of medical conditions such as cancer, thanks to the use of exosome capture beads and other methods of exosome detection.

In this sense, flow cytometry analysis of pre-enriched exosomes in plasma and other types of samples become very interesting, and we can do this analysis because exosomes can be captured by targeting these markers using magnetic beads.

Furthermore, the isolation and characterization of these exosomes from human fluids and cell culture media represent a huge opportunity because it has been demonstrated that this kind of analysis provide very useful information for early disease detection, monitoring and development of effective treatments.

On the other hand, exosome biology can be also very important in research about the use of these nanovesicles in regenerative medicine and vaccination research and increase the efficacy of therapeutic antibodies.

▌What are exosome capture beads?

 Magnetic capture beads are tools that contain very small (20 to 30 nm) particles of iron oxides, such as magnetite (Fe3O4), which give them superparamagnetic properties. Furthermore, these superparamagnetic beads exhibit magnetic behavior only in the presence of an external magnetic field.

In this sense, magnetic capture beads are very versatile tools for the exosome isolation and downstream analysis. These capture beads are usually used for different molecular biology applications such as next-generation sequencing (NGS), polymerase chain reaction (PCR) and many others. Which make these capture beads interesting is that this method ensures accurate and reproducible results.

▌Why to use exosome capture beads to characterize and detect exosomes?

Until now, the most popular exosome isolation protocols has been differential centrifugation with a final ultracentrifugation step, density gradient or cushion, size exclusion, and precipitation.

However, differential ultracentrifugation is time consuming, requires expensive equipment, and cannot discriminate between exosome subpopulations or other particles with similar size and density such as protein aggregates, lipids, and miscellaneous nucleic acid complexes.

In this sense, in order to obtain ultrapure exosomes or to isolate potential exosome subpopulations, an immunomagnetic isolation strategy can be applied by targeting exosomal surface markers.

This is the reason why magnetic capture beads have become more and more a popular versatile alternative tool in molecular biology for easy and effective isolation of biomolecule such as exosomes.

Their previously mentioned paramagnetic property makes it possible to separate beads in suspension, along with other particles they may be bounded to. Since they do not attract each other outside of a magnetic field, they can be used without thinking about undesirable agglomerations.

This simplicity in use allows the magnetic beads to be easy to automate and well suited for a wide range of applications by flow cytometry.

▌What is magnetic separation?

In molecular biology magnetic separation uses supermagnetic capture beads in order to provide a simple and reliable method of separating and accurately labeling enriched and unenriched exosomes in biological and other types of samples.

This way, with proper coating of the bead surface and optimized conditions, the target molecule will selectively bind to the beads, leaving pollutants in solution. This method is applied in order to use the purified targeted molecule (exosomes in this case) directly in analyses and applications.

The main advantages of using magnetic capture beads for magnetic separation is firstly that these beads allow biomolecules such as exosomes can be directedly isolated from a wide variety of different types of samples with minimal processing; and secondly, they have allowed the development of  very versatile and simply methods based on these magnetic capture beads.

▌Why is important the accuracy of exosome labeling for selective isolation?

The investigation of exosomes, as well as their tracking, delivery, and targeting via exosome labeling make it possible to determine their physiological functions and to exploit information for exosome-based research. In order to isolate the exosomes, they need to be labeled, so they can be differentiated from other particles in the process.

In this sense, some of the different ways of labeling the exosomes have been addressed so far, including exosome fluorescent labeling.

Fluorescent dyes can directly label proteins on exosomes. Therefore, fluorescent dye-labeled exosomes can be used to visualize and track specific components in isolated exosomes or to supervise the delivery of exosome cargo to cells in real time.

▌What is the efficiency of the immunomagnetic separation process?

Arrays based on immunomagnetic separation are a very useful tool for EV research.

As a proof of this, in a typical discovery-oriented experiment, a biological sample was analyzed by separating, quantifying and identifying as many exosomes as possible, often emphasizing those exosomes with an altered abundance relative to a reference sample.

At the end of this experiment, it was demonstrated that while microarray technology undoubtedly has an important role to play in discovery-oriented proteomics, it was clear that it will also be particularly well suited for EV research.

In this sense, the method is a sandwich immunoassay consisting of beads that differ in size or fluorescence intensity, resulting in color-coded beads conjugated with capture antibodies that are used for multiplexed immunoassays that differ from each other by their light scattering characteristics.

Some of the interesting aspects of bead-based arrays are fluid-phase kinetics, which is faster than flat-array solid-phase kinetics, higher precision, due to measurements of hundreds of beads for each analyte, and easy customization.

If you want to find out more about immunoaffinity bead-based isolation have a look at this article about commonly used exosome isolation methods.

▌How Exosome detection capture kits work for exosome detection by flow cytometry? 

Flow cytometry is a perfectly adapted technique for immunophenotyping assays, since at present the vast majority of cytometers allow a multiparametric detection (8–50 fluorescence channels) enough to identify the different exosomal subpopulations. For immunophenotyping characterization it will be necessary to have available conjugated antibodies with different fluorochromes, which will work as detector antibodies in the assay.

On the one hand, in the context of the use of cytometry as a suitable technology for liquid biopsy assays based on tumor exosomes, it should be noted that this methodology allow users to analyze multiple tumor exosomes simultaneously.

In this sense, Immunostep have developed a platform of methods based on magnetic capture beads by flow cytometry. This line of products are open and configurable for most commercially available cytometers so that clinicians can build their own panels or combinations, depending on the type of samples.

Moreover, these assays allow the characterization of captured exosome population through antibody coated beads against common exosome markers, such as tetraspanins (CD9, CD63 or CD81), allowing phenotype analysis of bulk exosome captured.

Furthermore, something that can happen is the loss of exosomes in the pre-enrichment steps, that is why, Immunostep offers capture beads that allow specific exosome isolation from different purification techniques.

▌How do I use Immunostep capture magnetic beads in flow cytometry?

Immunostep Exosome capture beads are able to isolate exosomes from biological fluids without previous enrichment procedures, allowing specific exosome isolation from different purification techniques. Furthermore, they are fully compatible with downstream analysis (WB, mRNA, miRNA…). You can access to the full information about these exosome capture beads in the technical data sheet. However, here we describe the full protocol to isolate exosomes and stain exosomes by fly cytometry:

►Isolate the exosomes:

1. Resuspend the capture beads by vortex for approximately 20 seconds
2. Add 50μL of the capture bead to each 12x75mm Polystyrene Round Bottom tube (cyto- meter tube). If an isotypic control needs to be added, please, do it at this point.
3. Add 50μL of sample previously prepared according to “Sample Preparation” to the appropriate tubes. Mix the reactions gently by pipetting up and down several times with a pipette and vortexing for few seconds.
4. Incubate in the dark overnight at room temperature (RT). NO STIRRING. After incubation if only exosome isolation is needed, without FACS analysis, go directly to step 12.

►Stain exosomes for flow cytometry

5. After overnight incubation, add the suggested volume indicated of the primary detec- tion antibody (not supplied. Visit our website: www.immunostep.com) to the bead-bound exosomes tube. Mix gently by pipetting and/or by tapping. It is advisable to prepare an additional tube with the appropriate isotype control or without exosomes, for background determination.
6. Incubate in the dark 60 minutes at 2-8oC, without stirring.
7. Wash the sample (bead-bound exosomes) by adding 1 ml of assay buffer 1X.
8. Collect the magnetic beads by placing tubes on a magnetic rack and incubate 5 minu- tes or by centrifugation at 2500xg for 5 minutes. Remove supernatant from tubes by hand-decanting in the case of using the magnetic rack (Fig. 3) or by aspiration. Take care not to disturb the microspheres, and make sure not to leave more than 100ul of super- natant in the tube.
9. Go to step 13 for direct stain protocol (using direct detection antibody labeled with fluorochrome) for indirect stain protocol go to next step (10).
10. Add 5μL of the secondary detection reagent (not supplied) t to each tube. Mix the reactions gently by pipetting up and down several times with a micropipette.
11. Incubate in the dark 30 minutes at 2-8oC, without stirring.
12. Wash the sample (bead-bound exosomes) by adding 1 ml of PBS-BSA 1% pH 7.4.
13. Collect the magnetic beads by placing tubes on a magnetic rack and incubate 5 minutes or by centrifugation at 2500xg for 5 minutes. Remove supernatant from tubes by hand-decanting decanting in the case of using the magnetic rack (Fig. 3) or by aspiration. Take care not to disturb the microspheres, and make sure not to leave more than 100 ul of supernatant in the tube. For exosome magnetic isolation without FACS analysis, protocol ends her, on the contrary, continue with next step.
14. Resuspend the sample in 350μLPBS-BSA 1% pH 7.4. and Acquire on a flow cytometer or store in the dark max up to 2 hours at 2-8oC, until the analysis is carried out.

►References

1. Jara-Acevedo R, Campos-Silva C, Valés-Gómez M, Yáñez-Mó M, Suárez H, Fuentes M. Exosome beads array for multiplexed phenotyping in cancer. J Proteomics. 2019; Apr 30;198:87-97.
2. Oksvold MP, Neurauter A, Pedersen KW. Magnetic bead-based isolation of exosomes. Methods Mol Biol. 2015;1218:465-481. doi:10.1007/978-1-4939-1538-5_27
3. https://experiments.springernature.com/articles/10.1007/978-1-4939-1538-5_27
4. https://www.cytivalifesciences.com/en/us/solutions/genomics/knowledge-center/magnetic-bead-guide/magnetic-bead-basics#:~:text=Magnetic%20beads%20(or%20superparamagnetic%20particles,which%20give%20them%20superparamagnetic%20properties.
5. https://expert.cheekyscientist.com/essential-beads-for-flow-cytometry-experiments/
6. Hu L, Chen X, Chen M, Fang J, Nie J, Dai H. Enrichment and detection of circulating tumor cells by immunomagnetic beads and flow cytometry. Biotechnol Lett. 2021;43(1):25-34. doi:10.1007/s10529-020-03007-8
7. Capture Beads for flow detection. Technical Data sheet. 2022. Available on: http://exosomes.imaisd.es/wp-content/uploads/2022/07/TDS-Exosome-Human-Capture-Beads-v5-Adri.pdf