Flow Cytometry Analysis of Inflammatory CNS Infiltrates

Flow cytometry was performed to immunophenotype inflammatory cells entering the CNS using established protocols (3 (link), 13 (link)). In brief, single cell suspensions were generated from tissue samples by grinding with frosted microscope slides. Immune cells were enriched by a 2-step Percoll cushion (90% and 63%) and cells were collected at the interface of the two Percoll layers. Before staining with the fluorescent antibodies, isolated cells were incubated with anti-CD16/32 Fc block (BD Biosciences, San Jose, CA) at a 1:200 dilution. Immunophenotyping was performed using either Rat anti-mouse IgG or Armenian hamster anti-mouse IgG antibodies for the following cell surface markers: F4/80 (Serotec, Raleigh, NC), MHV S510-tetramer (NIH), MHV M133-Tetramer (NIH) and CD4, CD8, Ly6g, CD11b, IFN-γ (BD Biosciences, San Jose, CA) and Ly6c (eBioscience, San Diego, CA). The following flow cytometric gating strategies for employed for immunophenotyping inflammatory infiltrates in the brain: neutrophils (CD45^hiCD11b+ Ly6G+); monocytes (CD45^hiCD11b+Ly6C+Ly6G−, macrophages (CD45^hiCD11b+F4/80+); microglia (CD45^lo, CD11b+, F4/80^lo); M133-specific CD4+ T cells (CD45^hi, CD4+, M133-tetramer+); S510-specific CD8+ T cells (CD45^hi, CD8+, S510-tetramer+).