Anti cd45.1 clone a20

Twelve to 24 h prior to infection, we transferred 2 × 10⁵ CD45.1⁺ OT-I cells (purified as above) i.v. into C57Bl/6 (CD45.2⁺) mice. For analysis of IFN-γ production, mice were infected i.p. with ~1 × 10⁸ TCID₅₀ of each recombinant. Spleens or peritoneal exudate cells (PECs) were harvested at 7 days PI, homogenized, and cells resuspended in RPMI-10 + 10 mM Hepes buffer and plated at 2 × 10⁶ cells/well in U-bottom, 96-well plates along with SIINFEKL or an irrelevant control peptide (SSIEFARL) at a final concentration of 100 nM. Cells and peptide were incubated for 4 h. at 37°C in the presence of 10 μg/ml brefeldin A (Sigma-Aldrich) to allow IFN-γ accumulation. For dead cell exclusion, cells were treated with ethidium monoazide (Invitrogen) before washing, incubating with Fc block (antibody 2.4G2 produced in house), and staining with anti-CD8 (clone 53–6.7) and anti-CD45.1 (clone A20; eBioscience). After staining, cells were washed and fixed at room temperature for 15 min with 1% paraformaldehyde. Cells were incubated overnight at 4°C with Alexa Fluor 647 anti–IFN-γ (clone XMG1.2, eBioscience) diluted in PBS containing 0.5% saponin (EMD Biosciences). Cells were analyzed on a BD LSR II and results analyzed using FlowJo.