Generating Plasma Membrane Vesicles from Bone Marrow Stem Cells
Transcrição
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.
