Anti cd8 pb

Groups of mice were sacrificed at the following time points: uninfected (day 0) and post-infection (days 5 and 14). At the indicated time points, spleens were collected from all animals and single cell suspensions were prepared. Cells were stained with anti-CD4-PO (Invitrogen), anti-CD44-FITC and anti-Thy1.1-PerCP (all from BD Pharmingen), anti-CD8-PB, anti-PD-1-FITC, anti-BTLA-PE, and anti-2B4-allophycocyanin (all from eBioscience).
For intracellular cytokine staining, single-cell suspensions of splenocytes were plated in a 96-well plate (1×10⁶ cells per well) in culture medium containing RPMI 1640 containing 10% FBS (Mediatech, Herndon, VA), 2 mM L-glutamine, 0.01 M HEPES buffer, 100 mg/ml gentamicin (Mediatech), and 5×10⁻⁵ M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Cells were incubated for 4 h in 10 nM OVA_257-264 (SIINFEKL; Emory University Microchemial Core Facility) and 10 mg/ml brefeldin A (Pharmingen). Following incubation, cells were stained with anti-CD4-PO (Invitrogen), anti-Thy1.1-PerCP (BD Pharmingen), and anti-CD8-PB (eBioscience) and processed using an intracellular staining kit (BD Biosciences) and stained with anti-IFN-γ-Alexa 700 and anti-IL-2-FITC (BD Biosciences). All samples were run on a LSRII flow cytometer (BD Biosciences), and data was analyzed using FlowJo 9.5 Software (Tree Star, San Carlos, CA).

STING KO mice and wild-type littermates were immunized as described above. Spleens were removed 7 days after primary immunization. Isolated splenocytes were directly used for staining with B8- or OVA-reactive tetramers. Briefly, splenocytes were washed with PBS and dead cells excluded by viability dye staining (eBioscience^TM Fixable Viability Dye eFluor^TM 506). Thereafter, cells were washed twice with staining buffer (1% BSA, 0.02% NaN₃ in PBS) and Fc-receptors blocked using CD16/32 antibodies (Fc-Block^TM, BD Biosciences). After washing, cells were incubated with B8- and OVA-reactive tetramers for 15 min and incubated in the dark on ice. Anti-CD8-PB, -CD127-APC, and -CD62L-PE/Cy7 (all eBioscience) antibodies were added to the tetramer-stained cells and incubated for additional 20 min in the dark on ice. Samples were analyzed by flow cytometry using BD FACS Canto II (BD Biosciences, Heidelberg, Germany).