Isolation of Phagosomes Containing Magnetic Bead-Tagged Bacteria from Infected Macrophages

Take a macrophage monolayer in phagosome isolation buffer supplemented with a reducing agent, enzyme inhibitors, and a cytoskeleton-disrupting compound.

These macrophages are pre-infected with bacteria tagged with biotin and streptavidin-conjugated magnetic beads.

During incubation, the reducing agent prevents protein oxidation, while enzyme inhibitors block protease activity, preserving phagosomal proteins. The cytoskeleton-disrupting compound destabilizes the membrane, aiding in cell rupture.

Replace with a buffer containing osmotic stabilizers to preserve the phagosome morphology.

Scrape the cells and collect the macrophage suspension in a pre-chilled tube.

Pass the suspension repeatedly through a needle to completely rupture the macrophage membrane while maintaining phagosome integrity.

Place the suspension on a magnetic rack. Under the magnetic field, phagosomes containing the magnetic bead-tagged bacteria attach to the tube wall.

Remove the supernatant and resuspend the phagosomes in fresh buffer.

Repeat magnetic separation and discard the debris-containing supernatant.

Resuspend the isolated phagosomes containing bacteria in buffer for downstream analysis.

First, prepare the volume of