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 a sterile 60-millimeter Petri dish containing 5 milliliters of PBS supplemented with 10% fetal bovine serum, and use a sterile 3 milliliter syringe plunger to disaggregate the tissue into a single cell suspension. Transfer the cells to a 15 milliliter centrifuge tube and collect them by centrifugation. Resuspend the pellet in 1 milliliter of red blood cell lysis buffer per 3 to 4 spleens.

After 5 minutes at room temperature, wash the spleens in PBS and resuspend the pellet in 250 microliters of PBS supplemented with bovine serum albumin and 2 millimolar EDTA for counting. Next, isolate the Ly6 B2 positive cells with the appropriate immunomagnetic beads according to manufacturer's protocol and seed the enriched Ly6 B2 positive cells at a 1 times 10 to the 5 cells per 100 microliters of complete medium per well density in a black or white 96-well plate. Prepare the bacterial inoculum at the desired multiplicity of infection in a final volume of 100 microliters per well and add 100 microliters of bacterial inoculum or medium alone to each well, for a 1 hour incubation in the cell culture incubator.

At the end of the incubation, replace the medium with 200 microliters of fresh complete medium supplemented with 100 micrograms per milliliter of gentamicin to remove any remaining extracellular bacteria and return the cells to the cell culture incubator for an additional 2 hours. At the end of the incubation and at each experimental time point thereafter, use a plate reader to measure the luminescence in each well of the lidded culture plate before returning the plate to the cell culture incubator until the next reading.