The overall goal of this procedure is to use magnetic particles or MNPs to identify multiple antigens on the surface of single virions or single extracellular vesicles with a conventional flow cytometer. This method can help us as the nature of the relationship between the phenotype and function of vesicles and viruses in human diseases, in particular, in viral infection. The main advantage of this technique is that it permits the analysis of single virions or single extracellular vesicles instead of in-bulk analysis in which the individual properties of these particles are lost.

This method can provide insight into the proper test of viral particles, but it can also be applied to other systems provided appropriate antibodies are available. Generally, individuals new to this method will struggle because all of the steps must be verified and checked before the analysis including the proper set-up of the flow cytometer. Begin by combining 60 microliters of MNPs per condition, and five microliters of labeled FAB fragments specific for the isotype of the capture antibody of interest in a 1.5 milliliter microcentrifuge tube.

Incubate the solution at room temperature for 30 minutes with continuous mixing. Then prewet a 100K concentrator with 300 microliters of PBS and spin the concentrator in the microcentrifuge. At the end of centrifugation, purify the labeled complexes on a 100K column followed by another microcentrifugation resuspending the pellet in 200 microliters of fresh PBS.

To capture the particles of interest, incubate the antibody labeled MNPs with the particle of interest for the appropriate incubation period and temperature. Next, block any nonspecific FC binding with the appropriate species of IGG antibody, gently vortex and incubate the particles solution for three to five minutes at room temperature. Then add the manufacturer recommended concentration of each detection antibody to the particles or isotype control antibodies to the isotype control sample for an additional 20 minutes incubation at room temperature.

To separate the MNP captured particles from the unbound antibody, first place a magnetic separation column in a separator magnet and prewet the column with 500 microliters of wash buffer. Allow the wash buffer to flow through the column, then add the MNP particle complexes to the column collecting the flowthrough in a waste container. When all of the complex solution has passed through the column, wash the column three times with 500 microliters of wash buffer.

Then transfer the column to a 12 by 75 millimeter tube. After three minutes, add 200 microliters of PBS, allowing the beads to elute by gravity. When all of the PBS has passed through the column, add 200 microliters of fresh PBS to the tube and fix the particles with 200 microliters of 4%paraformaldehyde.

Next, load 0.22 micron filtered PBS onto a flow cytometer and adjust the PMT voltage on the cytometer until the filtered PBS shows zero to very few events. Then run the MNP captured particles using a low flow with a 0.04 micron in-line filter for sheath fluid placed in series behind the standard 0.2 micron sheath filter to further eliminate any false events. With flow virometry, it is now possible to visualize cellular antigens on individual viral particles.

For example, in this experiment a high heterogeneity of HIV-1 virions was observed for the presence of LFA-1 and HLA-DR with viral particle capture that was dependent on the cells that replicated the virus. Dengue virus maturation is associated with the expression of precursor membrane proteins on the virions which can be distinguished using flow virometry as just demonstrated. In this representative experiment, extracellular vesicles were captured from platelet poor plasma using various fluorescent MNP coupled antibodies to investigate different extracellular virus subsets in patients with acute coronary syndrome.

Flow virometry analysis determined that while overall extracellular vesicle numbers were mostly increased in acute coronary syndrome patients compared to healthy donor controls, the magnitude of the increase varied between the different extracellular vesicle subpopulations. Once mastered, this technique can completed in two hours if it is performed properly. While attempting this procedure, it's important to remember to properly set up your flow cytometer before beginning the analysis.

The development of this technique opened a way for researchers in the field of virology and extracellular biology to investigate the roles of these particles in normal and pathologic processes in in-vitro system and in human and animal samples. Following this procedure, other methods like PCR or mass spectrometry can be performed to answer additional questions like what proteins or RNA or DNA molecules do specifically captured EVs or viruses carry. I hope that after watching this video you have a good understanding of how to analyze the antigenic composition of individual virions or individual extracellular vesicles.