Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Transcript

Extracellular Vesicles are increasingly being used as a diagnostic biomarker and potential therapeutic tool in many field. Flow Cytometric is the most straight forward analytic method for characterizing by suit faced markers this protocol can be used in analyzing an assized particle, is fast, and is applicable to conventional seismometers without the need for special setting or the dedicated instrument. Although this protocol is used to categorize Extracellular Vesicles from said condition and medium, it can be expanded towards categorization of blood arrived AVs, to replace invasive tissue biopsies in patients.

For researchers new to the protocol, it is critical to perform steps described in the set-up method section, and to closely follow the demonstrated getting strategies Begin by coating 55 centimeter squared Petri-dishes with 02%porcine skin gelatin, in PBS. After 15 minutes, wash the dish and plate 4.4 times 10 to the fifth cardiac progenitor cells onto each dish in 7 milliliters of Iscove's Modified Dulbecco's Medium, or IMDM, supplemented with 20%fetal bovine serum, and 1%penicillin and streptomycin for their incubation at 37 degrees Celsius and 5%carbon dioxide. When the cells reach about 80%confluency, wash the cultures two times with Dulbecco's PBS without calcium and magnesium and feed the cells with 10 milileters of serum free exosome production medium.

After 7 days, pool the conditioned medium from each plate, into a single polypropylene centrifuge tube and centrifuge the medium to remove any cell debris. Transfer the supernatent into a 100 kilodalton centrifuge filter tube, And concentrate the cleared, conditioned, medium with an additional centrifugation. Transfer the concentrate into a micro centrifuge tube for a final benchtop centrifugation and add the supernatent into a polycarbonate thick wall micro centrifuge tube.

Fill the tube with PBS, to a final volume of 3.2 milileters, and load the sample into a titanium fixed angeled rotor. Then load the rotor into a tabletop ultracentrifuge for ultracentrifugation, and resuspend the pellet in 100 microliters of fresh PBS. For nanoparticle tracking and analysis quantification, dilute 1 microliter of the ultracentrifuged sample, in 999 microliters of PBS, and load the sample into a 1 milileter syringe without bubbles.

Load the syringe into the inlet port of the examination chamber, and turn on the laser. Use the capture button to open the camera, and adjust the focus. Then record at least three different frames of 60 seconds each, and use the batch process option in the software, to analyze the three different acquisitions.

To prepare the sample for acquisition, first add the appropriate experimental monoclonal antibody conjugated capture beads at a 1:1:1 ratio, into a micro centrifuge tube, and vortex the resulting capture bead pool. Next, add the appropriate volume of sample to 1 microliter of capture bead pool to generate a 1 times 10 to the 8 particles plus 1.2 times 10 to the 5th beads per test concentration. Add 1 microliter of beads to a tube labeled beads as the negative control, and adjust the volume in each tube to 100 microliters with fresh PBS.

Then place the tubes in a thermo mixer, with shaking, at 400 rotations per minute, and 4 to 10 Celsius, overnight. The next day, transfer the samples to a round bottom 96 well plate, and add the appropriate florescence conjugated antibodies to each well. After a 1 hour incubation at 4 to 10 degrees Celsius, add 100 microliters of PBS to each well, and open the acquisition software.

Select cytometer and system start up program, to start the instrument and open a new experiment. Create an experimental template, and select plate and add plate. Select the position of the samples on the plate, and click set as sample well.

Enter the sample name in the naming rules, open the channel tab, and select the appropriate florescence signal channels. Now load the plate into the instrument. And click dot plot, to create a new forward scatter area versus side scatter area dot plot.

Select initialize to start the laser and the fluidics and click run to start the acquisition. Adjust the scale to show the population in the middle of the forward versus side scatter plot, and draw a beads gate around the whole sample population to exclude the debris. Click stop and dot plot to create a forward scatter height versus forward scatter area dot plot, and gate the sample on the beads population.

Draw a new gate around the bigger population and name it Singlets, and click dot plot to create one fluorescent signal versus side scatter area dot plot, per experimental fluorophore used. Gate the new dot plot on the Singlets. Then select the number of events to record, and select record to start the experimental acquisition.

At the end of the analysis, load the acquisition files into the appropriate analysis software, and open a new protocol. Create dot plots for each file as just demonstrated, and set all of the scatter axis to linear scale, and all of the florescent axis to log scale. Then show the florescence geometric mean in the relevant signals channels, to calculate the full change in mean florescence intensity, in the different extracellular vesicle preparations.

After testing different conditions to set the smallest total amount of extracellular vesicles to reach the early exponential phase of a mean florescence intensity curve, the minimum number of particles was determined to be 1 times 10 to the 8 particles per staining. The optimal concentration of antibody for 1 times 10 to the 8 particles to achieve the highest signal without nonspecific antibody binding, was determined to be 10 micrograms per milliliter for both anti CD9_FITC and anti CD63_PE antibodies and five micrograms per milliliter for the anti CD81_PE antibody. To confirm the suitability of the method, for analyzing cup shaped extracellular vesicles, and not membrane debris, the extracellular vesicles were sonicated, resulting in a decrease in mean florescence intensity, after as little as 30 seconds of sonication, with no florescence detected at all after one minute of mechanical disruption.

X's own purification from cardiac progenitor cells as demonstrated, yields extracellular vesicles, expressing low levels of CD9 and high levels of CD63 and CD81 from both cell populations. Furthermore, while X's own surface markers are clearly identifiable without ultracentrifugation, this enrichment step greatly enhances the mean florescence intensity of these markers. When working with extracellular vesicles, there's more careless nano particle pellets can be easily missed.

Therefore, it is essential to follow all the step as demonstrated in the protocol. Using extracellular vesicles as a biomarker is now one of the most emerging field research. And soon, may be translated into the clinical diagnostic used as an alternative screening tool.

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Journal (Biology)

Journal (Biology)

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

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