Cd8β yts156

Lymphocytes were isolated from peripheral tissues as previously described (23 (link)). Isolated lymphocytes were stimulated with LCMV gp_33–41 peptide (1µg/ml) for 4hrs at 37°C and cytokine secretion was evaluated as described earlier (35 (link)). Single cell suspensions were stained with antibodies against CD69 (H1.2F3), CD44 (IM7), Thy1.1 (OX-7), CD62L (MEL-14) from Biolegend, CD8a (53–6.7), CD103 (M290), CD45.1 (A20), CD45.2 (104), IFNγ (XMG1.2), TNFα (MP6-XT22), IL2 (JES6-5H4) from eBioscience and BrdU (B44) from BDBiosciences. Samples were acquired on a Fortessa flow cytometer (BD Biosciences). Immunofluorescence was performed as described(36 (link)). Antibodies against Thy1.1 (OX-7), CD45.1 (A20), and CD8β (YTS156.7.7) from Biolegend were used to stain sections.
For in situ tetramer staining, mouse female reproductive tracts were manually cut into ~500 µm sections using a surgical blade. Tissue pieces were incubated with PE conjuagted tetramers in a 2% fetal bovine serum in phosphate buffered saline (PBS) overnight at 4°C. The next day, tissues were washed with ice cold PBS three times on ice. Tissue pieces were then fixed in 2% paraformaldehyde in PBS for 2 hours, then washed in PBS and then placed in a 30% sucrose PBS solution overnight. The next day tissue pieces were frozen in OCT.

Leukocytes were stained for CD8β (YTS156.7.7; Biolegend), CD4 (RM4–5; BD Pharmingen), CD44 (IM7; eBiosciences), IFNγ (XMG1.2; BD Pharmingen). For intracellular cytokine staining, splenocytes from recipient mice were incubated with LPS-matured, irradiated syngeneic (H2^b) or allogeneic (H2^d) stimulator cells and assessed for intracellular IFNγ production as described previously (20 (link)). Samples were analyzed on an LSRII flow cytometer (Becton Dickinson).