The overall goal of this protocol is to isolate and characterize neutrophil-derived microparticles for functional studies. This method can help answer key questions about the role of exo-cell vesicles, or microparticles, in cell-cell communication in different physiological processes including cancer, inflammation, and wound healing. The main advantages of this technique are that it is relatively quick, cost effective, and that it allows the valuation of the p`henotypic composition of microparticles, or micro-vesicles, in functional assets.
Demonstrating the procedure will be Ariel Finkielsztein, a postdoctoral fellow in my laboratory, and Joseph Lee, an undergraduate in my laboratory. For density gradient separation of murine bone marrow polymorphonuclear, or PMN cells, first slowly layer three milliliters of freshly-prepared, room temperature, 1.077 grams per milliliter solution over three milliliters of freshly-prepared, room temperature, 1.119 grams per milliliter density solution in one 15 milliliter conical tube per bone marrow cell samples. Next overlay one milliliter of freshly isolated bone marrow cells in ice cold PBS onto the top density gradient layer of each tube, and separate the cells by density gradient centrifugation.
At the end of the separation, carefully remove the mononuclear cell layers at the interfaces between the PBS and the upper density gradient layers in each tube without disturbing the PMN bands. Using a transfer pipette, collect the PMN layers into one new 15 milliliter tube per sample, and bring the final volume in each tube up to 15 milliliters with 0.1 micron pour-size filtered PBS. Pellet the PMNs by centrifugation, followed by a second centrifugation in two milliliters of fresh-filtered PBS per tube.
Then re-suspend the pellets in one milliliter of fresh-filtered PBS for counting. After counting, centrifuge the cells again, and re-suspend the pellet in 100 microliters of 0.1 micron pour-size filtered HBSS, supplemented with the stimulant of interest per 10 million cells. Incubate for 20 to 30 minutes at 37 degrees Celsius.
At the end of the stimulation, collect the cells by centrifugation and transfer the cell-free supernatants into one new, 1.5 milliter micro-centrifuge tube per sample. Centrifuge the supernatants to remove any cell debris, and transfer the cleared supernatants into new ultra-centrifuge tubes, then ultra-centrifuge the supernatants, and store the pelleted microparticles in paraffin-sealed ultra-centrifuge tubes at 80 degrees Celsius until further experimental analysis. To administer the microparticles into the mouse colon, first place a fully anesthetized mouse in the prone position, and use biopsy forceps and an endoscope equipped with a high-resolution camera to generate three to five superficial wounds along the dorsal side of the colon.
Return the mouse to its cage with monitoring until full recovery. 24 hours later, use the endoscope to acquire images of the inflicted wounds under anesthesia. Then use a colonoscopy-based micro-injection system to administer an experimental volume of PMN microparticles re-suspended in 100 microliters of HBSS directly into each wound site.
The size heterogeneity of the PMN microparticles can be assessed by comparing the microparticles to known-sized flow cytometry beads. As demonstrated, the expression of proteins of interest by the microparticles can also be examined by flow cytometry. In this experiment, the expression of key inflammatory and anti-inflammatory molecules by activator-stimulated PMN-derived microparticles was assessed by immunoblot.
The affects of PMN microparticles on epithelial cell healing can be monitored by image acquisition of scratch-wounded epithelial monolayers at pre-determined time points in vitro, as well as after biopsy forceps wounding in vivio. Once mastered, PMN-derived microparticles can be isolated within four hours, if the technique is performed properly. While attempting this procedure, it is important to remember that prior to stimulation, the PMN should be kept on ice to prevent premature activation and microparticles lost.
Following this procedure, flow cytometry, immunoblotting, and real-time proliferation reaction techniques can be used to define the microparticle content, under various stimulatory conditions, or from different cells of origin. This technique is useful for examining the role of microparticle contribution in the regulation of immune responses, barrier function, tissue injury and repair, as well as in cancer progression. After watching this video, you should have a good understanding of how to isolate and label murine microparticles and to use the microparticles in a functional wound-healing asset in vivo.
Do not forget that working with live animals can be hazardous, and that precautions suggest putting on the personal protective equipment and completing the appropriate safety trainings should always be undertaken before performing this procedure.
