JoVE Encyclopedia of Experiments
Immunology
0 views • 3:20 min • July 8th, 2025
Begin with a bone marrow stem cell culture.
Introduce the trypsin-EDTA buffer to detach the cells and break the cell clumps, forming a single-cell suspension.
Centrifuge, then remove the supernatant. Resuspend the pellet with a fresh medium.
Transfer the suspension into a syringe and connect it to a membrane filter unit.
Push the plunger and discard the filtrate-containing medium.
Detach the filter. Fill the syringe with a fresh medium. Connect the filter again, and rapidly push the plunger.
The pressure of the liquid forces the cell toward the filter pores.
This produces a spherical plasma membrane vesicle or PMV.
These vesicles contain cell components and organelles surrounded by the membrane.
Examine the filtrate under an inverted phase-contrast microscope to observe translucent spherical vesicles.
Dimensions of the vesicles match with the pore size of the filter membrane, confirming the generation of PMVs.
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-f
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