Immunophenotyping of JHMV-infected Mice

Immunophenotyping of immune cells present within brains and spinal cords of JHMV-infected mice at defined times post-infection (p.i.) was accomplished by homogenizing isolated tissue and generating single-cell suspensions for analysis by flow cytometry using previously described procedures [19 (link)–21 (link)]. In brief, isolated cells were stained with the following antibodies: APC-conjugated rat anti-mouse CD4 and a PE-conjugated tetramer specific for the CD4 immunodominant epitope present within the JHMV matrix (M) glycoprotein spanning amino acids 133-147 (M133-147 tetramer) to determine total and virus-specific CD4⁺ cells, respectively [19 (link)–21 (link)]; APC-conjugated rat anti-mouse CD8a and a PE-conjugated tetramer specific for the CD8 immunodominant epitope present in the spike (S) glycoprotein spanning amino acids 510-518 (S510518) to identify total and virus-specific CD8⁺ cells, respectively [19 (link)–21 (link)]. Tetramers were synthesized by the NIH tetramer core facility: APC-conjugated rat anti-mouse CD4 and PE-conjugated anti-CD25 to determine total T-regulatory cells and BV510-conjugated rat anti-mouse CD45 and FITC-conjugated anti-F4/80 to identify macrophages. Samples were analyzed using a BD LSR Fortessa X-20 flow cytometer and FlowJo software.