The overall goals of this method, are to characterize and study populations of plasma membrane-derived microvesicles in peripheral blood. Microvesicles from tumor patients, are proinvasive and functional assays. Therefore, further characterization will provide information on how these vesicles actually contribute to invasion and metastasis.
The main advantage of this technique is that it allows a relatively quick analysis of microvesicles in blood, without the need for sophisticated or expensive materials and equipment. After obtaining one or two tubes of blood from a volunteer into collection tubes containing EDTA, invert the tubes several times to guarantee efficient blood anticoagulation. Within a half hour of the blood draw, centrifuge the samples, and add a valve filter to the tubes to help separate the plasma from any remaining blood.
Transfer the plasma into a 15 milliliter tube for a second centrifugation. Harvest the supernatant and transfer it into a new tube. Add PBS if necessary.
Centrifuge the plasma containing supernatant diluted in PBS as necessary to collect the microvesicles. Then decant the supernatant and place the tubes upside down on a paper towel for three to five minutes. When all the remaining supernatant has soaked into the towel, remove any remaining plasma from the rim of the tubes, and resuspend the microvesicle pellet in one milliliter of PBS.
Transfer the solution into a 1.5 microcentrifuge tube. Then after centrifugation, aspirate the supernatant, and resuspend the pellet in 50 to 500 microliters of PBS, depending on the size of the pellet. Then determine the microvesicle protein concentration with a protein assay.
To characterize the microvesicle's by flow cytometry, transfer 15 microliters of PBS, supplemented with 1%vesicle-depleted FCS into a flow cytometry tube, followed by three to five micrograms of microvesicles suspended in PBS. Incubate the sample for 30 minutes at room temperature to block any nonspecific binding on the microvesicle surface. Then add the fluorescently labeled antibody of interest for a 20-minute room temperature incubation in the dark.
Next, dilute the sample in 250 microliters of fresh PBS, and load the microvesicles onto the flow cytometer. Reduce the threshold of the flow cytometer to the lowest possible value, and search the microvesicle population using a forward versus side scatter plot in a logorithmic scale. Then make a gate around the microvesicle population, and evaluate the fluorescent signal in a corresponding histogram.
The microvesicle yield from donor blood samples can be assessed in the Lowry protein assay with typical yields between 10 to 30 micrograms of vesicle per millileter of blood, and a particle concentration ranging from 1.66 times 10 to the 9th, to 2.36 times 10 to the 10th, as determined by the interparticle tracking analysis. Further characterization of the microvesicles by transmission electron microscopy reveals a population of vesicles with a diameter of greater than 100 nanometers surrounded by a lipid bilayer that does not contain any cell organelles. Nanoparticle tracking analysis also confirms that the average size of the isolated microvesicles ranges from 100 to 600 nanometers with a mean of 201 nanometers.
Staining for typical microvesicle and exosome markers by western blotting demonstrates that the blood isolated microvesicles are typically positive for tubulant, with a weak expression of CD9 and CD81, while exosomes are generally negative for tubulant, and are enriched with the other two markers. Flow cytometry reveals a defined donor-derived vesicle population that can be gated using the same parameters used for cell culture supernatant isolated microvesicles. That is also clearly different than the background signal observed for the controlled PBS, supplemented with 1%vesicle-depleted FCS-only samples.
Microvesicle staining with established markers for different blood cell populations reveals the percentage of microvesicle subpopulations differs among blood donor samples with the majority of microvesicles appearing to be shed by platelets in all of the samples. Once mastered, the isolation of microvesicles and their subsequent characterization by flow cytometry can be performed in about 2-1/2 hours. Characterization of microvesicles subpopulations will help to identify a tumor signature in patients versus controls.
This may be useful as a biomarker for counter diagnostics and therapy.
