Magnetic Isolation of Microglia from Mouse Pup Brains
Transcrição
Microglia — the brain-resident macrophages — express CD11b, a cell-surface integrin.
To isolate microglia from mouse pup brains, take cerebrum fragments in dissociation tubes containing buffer with papain, a proteolytic enzyme, and deoxyribonuclease.
Place the tubes on a mechanical dissociator. During the run, the dissociator mechanically disrupts the tissue.
Papain digests the tissue’s extracellular matrix, releasing cells, including microglia, into suspension. Deoxyribonuclease degrades free DNA in suspension, preventing inefficient cell isolation.
Centrifuge. Complete the mechanical dissociation by pipetting. Transfer the suspension through a cell strainer to remove debris and cell clumps, and obtain a homogeneous single-cell population.
Incubate with superparamagnetic microbeads functionalized with anti-CD11b antibodies. The microbeads specifically bind to CD11b on cells, including microglia.
Post-incubation, centrifuge. Remove the supernatant containing unbound beads. Resuspend the cells in buffer. Load onto a column placed on a magnetic separator. The column comprises a matrix with ferromagnetic beads.
When placed on the separator, the spheres within the column amplify the magnetic field, retaining the microbead-bound CD11b+ cells within the column, while other cells flow through.
Remove from the separator and use buffer to elute the CD11b+ cells from the column. Centrifuge the suspension and resuspend the CD11b+ cells in a microglia medium.
The isolated cells are ready for further analysis.
