Generating Plasma Membrane Vesicles from Bone Marrow Stem Cells
Transcript
To generate the PMVs, first, culture mouse PMSCs in complete DMEM culture medium in one well of a six-well plate in a humidified incubator at 37 degrees Celsius and 5% carbon dioxide. When the cells have reached 90% confluency, rinse the well with 0.5 milliliters of calcium-free PBS, and detach the cells with 0.5 milliliters of 0.25% trypsin-EDTA.
After three minutes at 37 degrees Celsius, tap the plate with the palm of the hand a few times to facilitate cell dissociation and stop the digestion with 1 milliliter of culture medium. Transfer the cells into a 5-milliliter conical tube for centrifugation and resuspend the pellet in 4 milliliters of fresh culture medium. Then, divide the cells between two wells of a new six-well plate and return the plate to the cell culture incubator for 24 hours.
The next day, harvest the cells from one well by trypsin digestion, as just demonstrated, resuspending the pellet in 0.5 milliliters of fresh culture medium after collection by centrifugation. Load the cells into a 1-milliliter insulin syringe, and attach the syringe to a filter unit. Quickly depress the plunger to squeeze the cells through the filter and discard the extruded medium. Then, load and dispense 0.5 milliliters of extra culture medium through the filter, as just demonstrated, collecting the medium from the second extrusion.
Seed 150 microliters of the collected medium into a 35-milliliter glass bottom dish and allow the vesicles to settle for about 10 minutes. Then, confirm the presence of PMVs under an inverted phase-contrast microscope equipped with a CCD camera using a 20-times objective lens.
