Assessing the Survival of a Bacterial Pathogen in Mouse Peritoneal Macrophages

Take a culture of mouse peritoneal macrophages and introduce a bacterial pathogen.

Incubate to allow bacterial uptake through phagocytosis into the macrophages, where they alter intracellular degradation pathways and survive.

Remove the supernatant and wash with a buffer to eliminate extracellular bacteria.

Add a medium containing gentamicin antibiotic and incubate to remove any remaining extracellular bacteria.

Wash the infected macrophages and mechanically detach them. Collect the cells, centrifuge, and remove the supernatant.

Add a bacteriolytic enzyme and incubate to lyse any additional extracellular bacteria.

Centrifuge and discard the supernatant. Wash the cells, centrifuge, and remove the supernatant.

Resuspend and aliquot the cells. Add a detergent to lyse them, releasing intracellular bacteria. Serially dilute the lysate in buffer and drop the dilutions onto agar.

Incubate to allow bacterial colony formation. Count the colonies to assess bacterial survival within the macrophages.

Incubate the peritoneal macrophages with Staphylococcus aureus MRSA-252 for two hours. Remove the supernatant and wash the cells twice with PBS. Add 1 milliliter per well of complete DMEM medium containing