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 required phagosome isolation buffer A by adding DTT and Cytochalasin B just before use, and protease and phosphatase inhibitors as recommended by the manufacturer. Remove the medium from the infected cells at the desired time point and wash the cells with sterile PBS pre-warmed to room temperature.

Then add 750 microliters of phagosome isolation buffer A to the dish and incubate on ice for 20 minutes. Remember to rock the plate occasionally. Next, add 250 microliters of phagosome isolation buffer B and then rock the plates to ensure that the buffer completely covers the surface.

Gently scrape the cells using a rubber policeman to remove the cells from the dish and transfer them to a pre-chilled 1.5 milliliter tube. Then use a 1 milliliter syringe to pass the cell suspension through a 26-gauge needle at least 15 times. Next, place the cell suspension on the magnetic rack for 5 minutes.

Particles attached to the magnetic beads are phagosomes containing coated S.Typhimurium. Transfer the suspension, which contains the rest of the cellular components, into a new 1.5 milliliter tube labeled as Cytosol. Remove the tube containing isolated phagosomes from the rack and resuspend in 1 milliliter sterile PBS.

Place the tube back on the magnetic rack for 5 minutes and then remove the PBS. After repeating this washing with PBS once, remove the PBS and finally, resuspend S.Typhimurium containing phagosomes in the required buffer.