Microglia-CD8+ T Cell Interaction Assay

After animal sacrifice at defined times p.i. and leukocytes enriched on a Percoll gradient as previously described (Blanc et al., 2014 (link)). Cell were sorted at the University of Utah Flow Cytometry Core based using the following markers, CD45^lo, CD11b+, F4/80+. Microglia were then plated in a 96-well flat bottom plate coated with Poly-D-Lysine (Sigma Aldrich) and incubated with the S510 peptide overnight. Spleens were collected from MHV-DM infected SPF mice 7 D.P.I., viral-specific CD8 +T cells were sorted at the University of Utah flow cytometry core. T cells were stained using Far Red Cell Trace or CFSE (Thermo Fisher), CD8 +T cells were then co-cultured with microglia for 72 hr, samples were analyzed using BD LSRFortessa and FACSDiva software and data was measured using FlowJo.