In Vitro Stimulation of Mouse Splenocytes with Heat-Killed Listeria monocytogenes

Begin with a single-cell suspension of splenocytes, harvested from a Listeria monocytogenes-infected mouse.

The suspension contains immune cells, including antigen-presenting cells and T cells.

The cells are suspended in a culture medium containing serum proteins to support cell viability.

Now, seed the suspension into a multi-well plate containing heat-killed L. monocytogenes.

During incubation, antigen-presenting cells engulf and process the bacteria, then display the bacteria-derived antigens on MHC molecules.

T-cell receptors recognize and bind to these bacterial antigens on MHC,

This activates the T cells and triggers cytokine release.

Next, add a protein inhibitor of vesicular transport and incubate.

Inside T cells, the inhibitor blocks vesicular transport, preventing further cytokine release.

Collect the culture medium containing cytokines.

Add a buffer and collect the cells separately.

The cytokine-containing culture medium and harvested cells are ready for downstream analysis to assess the L. monocytogenes-induced cytokine production in T cells.

To measure CD4 and CD8 T cell interferon gamma responses, harvest the spleens from mice infected with two times ten to the fourth CFU of the