Flow Cytometry-based Sorting of Infected Cells

For flow cytometry-based sorting of infected cells, mice were infected with 10⁴ PFU MHV68-H2bYFP, a phenotypically wild-type virus that expresses eYFP under control of the H2b promoter [44 (link)]. At 16 dpi, splenocytes were prepared and blocked as described above. Cells were then stained with APC rat anti-mouse CD4 at 1:200 (BD Biosciences, 553051), APC rat anti-mouse CD8α at 1:200 (BD Biosciences, 553035), APC rat anti-mouse CD14 at 1:100 (BD Biosciences, 560634), and APC-Cy7 rat anti-mouse CD19 at 1:200 (BD Biosciences, 557655). Infected B cells (CD4^-CD8^-CD14^-CD19⁺YFP⁺) and non-infected B cells (CD4^-CD8^-CD14^-CD19⁺YFP^-) were sorted using a BD FACSAria II flow cytometer (BD Biosciences). Sorted cells were immediately subjected to RNA extraction using an RNAqueous-Micro kit (Ambion, AM1931) prior to qRT-PCR analyses.