Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

The overall goal of this procedure is to isolate and analyze extracellular vesicle circulation in the blood by two different methods. For the individual extracellular vesicle detection method. Platelet poor plasma or PPP is first isolated from a blood sample.

The PPP is then stained with the fluorochrome conjugated antibodies of interest. For the bead based detection method, the extracellular vesicles are incubated with beads, and then the bead bound vesicles are stained with the relevant fluorochrome conjugated antibodies. Ultimately, the circulating extracellular vesicle content can be determined by analysis of the samples by flow cytometry.

The main advantage of this technique over existing methods like electron microscopy or magnetic bead separation, is that it's much faster and allows for the assessment of the total extracellular escal population rather than being limited to specific subpopulations. Generally, individuals new to this method will struggle as the success of this method requires a strict adherence to the demonstrated maneuvers and cytometer setup parameters In order to produce high quality data with minimal day-to-day variation, Begin by centrifuging whole blood samples to separate the plasma from the Buffy coat and red cells. Next, transfer 1.2 milliliter aliquots of the plasma supernatant into 1.5 milliliter centrifuge tubes, taking care not to disturb the bottom layers containing the buffy coat and the red cells then centrifuge the supernatant again to remove any platelets and large cell fragments.

At the end of the spin, carefully transfer all but the last 200 microliters of the PPP samples to new tubes, taking care not to disturb the pellets. Then mix the plasma up and down several times and transfer 320 microliters of each sample to the top row of a 96 well plate. To stain the samples, combine the antibodies of interest for each of the three panels into individual zero point 22 micrometer, centrifugal filter tubes, and spin down the antibodies using a fixed angle, single speed centrifuge.

When all of the antibody has passed through the filter, add the appropriate amount of the mixture to each well of the 96 well plate as depicted in the figure. Next, use a multi-channel pipette to mix the PPP samples and then transfer 100 microliters of each sample to the antibodies in row two. Then mix the samples again, change the tips and transfer 100 microliters of sample to each of the next two rows of antibodies as appropriate.

For the experiment. Incubate the plate at four degrees Celsius after 30 minutes at 220 microliters of PBS per well to row six through eight inside a biological safety cabinet. Then using a width adjustable multichannel pipette, transfer the contents of each well to its corresponding centrifugal filter tube without changing the tips.

Use 200 microliters of PBS from one of the wash rows to rinse the wells from which the samples were just removed. Then transfer the rinse solution to the same filters to which the PPP samples were previously added, and close the centrifugal filters. Once all of the stained PPP samples have been transferred along with their rinse solutions, spin down the samples in a fixed rotor centrifuge.

After the spin, ensure that no liquid remains on top of the filters. Then resuspend the tops of the filters in 300 microliters of PBS and transfer the resuspended contents to pre-labeled tubes for immediate flow cytometric analysis. Washing the stained samples using centrifugal filters, enhances the separation between the background and positive marker signals.

It's important to note however, that some of the smaller extracellular vesicles, including exosomes, may be lost through the doors of the filter To analyze the samples, first, set the forward and side scatter voltage parameters to a log scale and select the lowest thresholds allowed by the cytometer for each parameter. Then while running a tube of zero point 22 micrometer, filtered PBS adjust these voltages to the highest values that exclude the majority of background noise. Next, run a tube containing a mixture of beads, one micrometer and smaller, and draw a gate around the bead population in a forward by-side scatter plot to capture all of the events under the one micrometer beads.

Now set the cytometers flow rate to low and use the beads to adjust the flow rate dial on the cytometer to the desired event rate. Then using a tube of rainbow fluorescent particles diluted in PBS acquire 5, 000 events, recording the mean intensity of values for the forward scatter side scatter, and each color channel. When all of the parameters are set, run each sample for exactly one or two minutes After the first reading for each tube has been completed, mix 20 microliters of 10%MP 40 into each sample, and reread the tubes for the same amount of time to allow the subtraction of the positive events detected in the lysed sample over an equal period of time.

To detect the extracellular vesicles by bead based capture, begin by washing uncoated six micrometer polystyrene beads two times with RPMI media, followed by Resus in two milliliters of the same. Next, add 6, 000 beads to each new fax tube to the negative control tube at 400 microliters of media to all the other tubes at 200 microliters of PPP or ultracentrifuge extracellular vesicles as appropriate, and 200 microliters of media. Adjust the final volume in each tube to 400 microliters.

With more media. Incubate the tubes overnight at four degrees Celsius on a shaker. The next morning, wash the beads with two milliliters of media.

Aspirate the supernatant and block any non-specific binding with 400 microliters of 5%bovine serum albumin place on a shaker at four degrees Celsius for three hours. After three hours, wash the beads in two more milliliters of media and resuspend the pellets in 100 microliters of fresh medium. Now filter the antibodies and add the appropriate volume of filtered antibody cocktail to each tube, and then after 30 minutes at four degrees Celsius, wash the beads in another two milliliters of media.

This time resuspend the pellets in 400 microliters of media and immediately analyze the samples by flow cytometry, adjusting the forward inside scatter parameters to the lowest threshold allowed by the flow cytometer as just demonstrated. Finally, gate on the singlet bead population and acquire 2000 events per sample. Both the individual and bead based detection methods can be used to detect the presence of the extracellular vesicles in the PPP samples as illustrated by these dot plots For the individual detection assay, the lysed control is used to set the gates for the corresponding non-listed sample.

The majority of events should fall within the extracellular vesicle gate. For example, in this figure, these bi parameter plots showed the detection of the markers, CD 1 0 8 A and CD 2 35 A, which are two red blood cell markers known to coexist on cells. As expected, over half of the positive events on the extracellular vesicles are positive for both of the markers, whereas in these by parameter plots, the extracellular vesicle expression of two markers that are known not to coexist on cells exhibit distinct separate positive populations using the bead based detection method.

No separation between the positive and negative populations exist and events appear in the double positive quadrants, even though they aren't normally found on the same cell types due to the fact that both types of extracellular vesicles will bind to a single bead. Therefore, data from this method are best analyzed using histograms overlaid with the negative control data, with an apparent shift in the marker expression observed in the beaded samples as compared to the controls Once mastered. This technique can be used to analyze 12 blood samples with three panels of antibodies in three to four hours with the individual detection method or in one and a half days by the bead method if it's performed properly.

While attempting this procedure, it's important to remember to be consistent in the treatment of each sample, ensure that the cytometer flow rate and fluorescent intensities have been adjusted correctly at the beginning of each experiment to match the previous day's experimental settings, especially when running multiple samples across multiple days.