In Vitro Evaluation of Bacterial Killing by Neonatal Mouse Splenic Phagocytes

Begin by mechanically dissociating neonatal mouse spleens to generate a single-cell suspension.

Transfer the suspension to a tube, centrifuge, and remove the supernatant.

Resuspend the pellet in lysis buffer to eliminate erythrocytes.

Add buffer, centrifuge again, and remove the supernatant.

Resuspend the cells in buffer and incubate with antibody-coated magnetic microbeads targeting a receptor expressed on neutrophils and inflammatory monocytes.

Centrifuge and remove the unbound beads. Add buffer, then pass the suspension through a ferromagnetic column and apply a magnetic field to retain the bead-bound cells.

Remove the magnetic field, elute the cells with buffer, and seed them into a multi-well plate.

Add increasing concentrations of luminescent bacteria to selected wells.

Incubate to allow neutrophils and monocytes to internalize the bacteria by phagocytosis.

Replace the media with antibiotic-containing media to eliminate non-internalized bacteria.

Record luminescence at regular intervals to assess bacterial internalization and subsequent killing induced by phagocytosis.

To perform an in vitro bacterial killing assay, place the uninfected spleen into a 40-micrometer nylon strainer within