Purification of CD31+ Endothelial Precursor Cells Using Magnetic-Activated Cell Sorting
Transcription
Take an adherent culture of endothelial progenitor cells or EPCs, precursors to brain microvascular endothelial cells.
The culture, obtained by differentiating pluripotent stem cells, comprises EPCs positive for the surface marker CD31 and contaminating CD31-negative cells.
Incubate with a dissociation reagent to detach the cells and filter to obtain a single-cell suspension.
Add a media to neutralize the enzymes, centrifuge to discard the supernatant, and resuspend the cells in a buffer.
Incubate with a blocking reagent to inhibit the cellular Fc receptors, preventing non-specific labeling.
Add a fluorophore-conjugated antibody specific for CD31 and incubate to label the EPCs.
Centrifuge and discard unbound antibodies. Incubate with antibody complexes containing one antibody that binds to the fluorophores.
Introduce polymer-coated magnetic nanoparticles and incubate, allowing the nanoparticles to bind to the complexes' second antibody.
Using a magnetic field, isolate the nanoparticle-bound cells.
Discard the suspension with unlabeled CD31-negative cells and resuspend the purified cells in a medium. The CD31-positive EPCs are ready for downstream assays.
