E fluor 660 conjugated anti gl7

Single cell suspensions of splenocytes were resuspended in PBS containing 2% fetal bovine serum and stained on ice for 20 minutes. Prior to staining, Fc receptors were blocked with anti-16/32 (BD Biosciences). Antibodies used were BV421 conjugated anti-Igλ (BD Biosciences), PE conjugated anti- Igκ (BD Biosciences), PE-Cy7 conjugated anti-CD95 (BD Biosciences), APC-Cy7 conjugated CD19 (BD Biosciences), PE conjugated CD138 (BD Biosciences), PerCP-Cy5.5 conjugated anti-CD3, CD4 and CD8 (Biolegend), Pac Blue conjugated anti-B220, and E-Fluor 660 conjugated anti-GL7 (eBioscience).
For each infection, a separate group of C57Bl6/J mice were infected in parallel with wild type MHV68. Gates for YFP+ populations were set based on the level of auto-fluorescence detected in the FITC channel from mice infected with wt virus. Single cells were sorted based on YFP and surface marker expression (Table 1). Cells were sorted into 96 well plates containing 4 ul of ice cold 0.5X PBS supplemented with 8U/ul of RNAsin (Promega). After sorting, plates were snap frozen in a dry ice/ethanol bath and stored at -80⁰C.

Single cell suspensions of splenocytes were resuspended in PBS containing 2% fetal bovine serum and stained on ice in the dark for 20 minutes. Antibodies used were PE conjugated anti-CD138, anti-CD86 and anti-Ly5.2 (BD Biosciences), PerCP conjugated anti-CD3, anti-CD4 and anti-CD8 (BD Biosciences) and anti-GL7 (eBioscience), PE-Cy7 conjugated anti-CD95 and anti-Ly5.1 (BD Biosciences), Pacific Blue conjugated anti-B220 (Biolegend), eFluor 660 conjugated anti-GL7 (eBioscience), and APC conjugated anti-CXCR4 and anti-CD138 (BD Biosciences), APC-Cy7 conjugated anti-CD8 (Biolegend) and APC-Cy7 conjugated anti-Ly5.1 (BD Biosciences). For each infection, C57Bl6/J mice were infected in parallel with wt MHV68. Gates for the YFP+ populations were set based on the level of auto-fluorescence detected in the FITC channel from mice infected with wt virus as shown in Fig 1A. Data was collected on an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., http://www.flowjo.com).